Qtl controlling sclerotinia stem rot resistance in soybean

ABSTRACT

Markers associated with  Sclerotinia  stem rot resistance are provided. Methods of identifying resistant, and susceptible plants, using the markers are provided. Methods for identifying and isolating QTL are a feature of the invention, as are QTL associated with  Sclerotinia  stem rot resistance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application under 35 U.S.C. §121 ofU.S. patent application Ser. No. 10/165,617, filed on Jun. 7, 2002,which claims the benefit of U.S. Provisional Patent Application Ser. No.60/297,044, filed Jun. 7, 2001, the disclosures of which areincorporated herein by reference in their entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to plant molecular biology. Morespecifically, it relates to nucleic acid markers for identification ofquantitative trait loci (QTL) associated with Sclerotinia stem rotresistance in dicotolydenous plants.

BACKGROUND OF THE INVENTION

Sclerotinia stem rot in soybean, also know as white mold, is caused bythe fungus pathogen Sclerotinia sclerotiorum. The disease was firstreported in Hungary in 1924, and has since been reported in many othercountries, including the USA. The disease has become increasinglyimportant in the USA since 1990. For example, Sclerotinia stem rotcaused an estimated yield loss of 6.4×10⁵ metric tons in the USA during1994 (Wrether et al. (1997) Plant Dis 81:107-110). Yield has been shownto be inversely correlated with the percent incidence of Sclerotiniastem rot. Yield loss has been estimated at 250 kg/ha for each 10%increment in diseased plants (Grau and Hartman (1999) Compendium ofSoybean Diseases, 4^(th) ed. APS Press, St. Paul, Minn., USA, pp.46-48).

S. sclerotiotum infects soybean plants in the form of ascospores thatland on flowers (Grau (1988) Am Phytopathol Soc, St. Paul, Minn., USA pp56-66). The ascospores germinate under adequate moisture condition anduse the flower petals as a nutrient base. Stem lesions originate at leafaxils where flowers are positioned and advance up and down the stem.Symptoms of the Sclerotinia stem rot begin to develop at growth stagesR2 and R3. Lesions are most frequently on the main stem and completelyencircle the stem and disrupt the transport of water, mineral nutrients,and photosynthates to developing pods. Pod development and pod fillabove stem lesions are reduced, resulting in yield reduction.

Soybean genotypes show large variations in resistance to Sclerotiniastem rot (Kim et al. (1999) Crop Sci 39:64-68). So far, no soybeangenotypes with complete resistance to S. sclerotiorum have beenidentified. The heritability of partial resistance, measured with adisease severity index (DSI), was estimated to be 0.59 with significantgenotype and environment interaction observed (Kim and Diers (2000)CropSci 40:55-61). Quantitative trait loci (“QTL”) analysis identified threeQTL explaining 10, 9, and 8% of the variability for DSI acrossenvironments and locating on linkage groups C2, K, and M. Two of theQTLs were also significantly associated with disease escape mechanismssuch as plant height, and date of flowering.

Breeding for white mold resistance via the traditional approach has beenvery difficult, due to the multigenic nature of this trait. What isneeded in the art is a means to identify genes conferring resistance towhite mold, using molecular markers. These markers can then be used totag the favorable alleles of these genes in segregating dicotpopulations and then employed to make selection for resistance moreeffective. The present invention provides this and other advantages.

SUMMARY OF THE INVENTION

The present invention provides methods and markers for identifyingQuantitative Trail Loci (“QTL”) associated with Sclerotinia stem rotresistance in soybeans and other plants. A first aspect of the inventionrelates to isolated or recombinant nucleic acids corresponding to markerloci useful for the identification and isolation (e.g., by positionalcloning) of QTL associated with Sclerotinia stem rot resistance inplants, particularly soybean. Such nucleic acid markers include:Satt155, SLS1C.L24, Sat_(—)129, Satt329, Satt556, P1694, PHP8701R,Satt311, PHP10118C, Satt231, P1047, A724_(—)1, Satt523, or SLS2C.F20,and homologous nucleic acids.

In another aspect, the invention relates to methods of identifying aSclerotinia stem rot resistant soybean. In some embodiments, the methodsinclude identifying a Sclerotinia stem rot resistant soybean byidentifying a QTL associated with resistance by detecting nucleic acidsin a soybean plant (or tissue of the soybean plant, e.g., a whole plant,a plant organ, a plant seed or a plant cell) that are genetically linkedto a locus proximal to the QTL. The detected nucleic acids typicallycorrespond to marker loci, such as the following simple sequence repeat(SSR) and restriction fragment length polymorphism (RFLP) marker loci:Satt155, SLS1C.L24, Sat_(—)129, Satt329, Satt556, P1694, PHP8701R,Satt311, PHP10118C, Satt231, P1047, A724_(—)1, Satt523, and/orSLS2C.F20. In an embodiment, linked pairs of nucleic acids are detected.For example, in one embodiment, nucleic acids that are homologous to thefollowing pairs of markers: a) Satt155 and SLS1C.L24; b) Sat_(—)129 andSatt329; c) Satt556 and P1694; d) PHP8701R and Satt311; e) PHP10118C andSatt231; f) P1047 and A724_(—)1; or g) Satt523 and SLS2C.F20, aredetected. In another embodiment, nucleic acids that are at least about80% identical to one of the above-listed markers are detected.

In some methods, plants identified as described above are crossed withplants lacking the detected nucleic acids. In some cases, Sclerotiniastem rot resistance is introgressed into progeny of the cross, i.e., thedetected nucleic acid is transmitted to at least a subportion of theprogeny derived from the cross.

Following identification of a QTL, nucleic acids corresponding to theQTL can be isolated by positional cloning, for example, by providing anucleic acid genetically linked to a locus homologous to one of themarkers listed above, and then cloning the nucleic acid. In someembodiments, the isolated or recombinant nucleic acid is a QTL localizedto a chromosome interval defined by loci homologous to markers a)Satt155 and SLS1C.L24; b) Sat_(—)129 and Satt329; c) Satt556 and P1694;d) PHP8701R and Satt311; e) PHP10118C and Satt231; f) P1047 andA724_(—)1; and g) Satt523 and SLS2C.F20.

Another aspect of the invention relates to compositions comprisingisolated or recombinant nucleic acids comprising QTL associated withSclerotinia stem rot resistance in plants, such as soybean. The isolatedor recombinant nucleic acids correspond to genomic loci localized withina chromosome interval flanked by loci having at least about 80% sequenceidentity to a pair of markers selected from among: a) Satt155 andSLS1C.L24; b) Sat_(—)129 and Satt329; c) Satt556 and P1694; d) PHP8701Rand Satt311; e) PHP10118C and Satt231; f) P1047 and A724_(—)1; or g)Satt523 and SLS2C.F20. In certain embodiments, the isolated orrecombinant nucleic acids correspond to loci localized to a chromosomeinterval flanked by marker loci having at least about 90%, (or at leastabout 95%, or at least about 98% or more) identity to one of theabove-listed pairs of markers. In some embodiments, marker loci areidentical to the above-listed sequences. In some embodiments, sequenceidentity is determined by the GAP algorithm under default parameters.

In preferred embodiments, the marker pair of (a) is on linkage group A1,(b) is on linkage group A2, (c) is on linkage group B2, (d) is onlinkage group D2, (e) is on linkage group E, (f) is on linkage group J,and/or (g) is on linkage group L.

Host cells, and transgenic plants (particularly soybean plants)comprising the isolated or recombinant nucleic acids described above arealso a feature of the invention.

Similarly, methods for making transgenic plants, particularly dicots,e.g., transgenic soybeans, are a feature of the invention. Such methodsinvolve introducing an isolated or recombinant nucleic acidcorresponding to a QTL associated with Sclerotinia stem rot resistance,as described above, into a plant cell, and growing the cell underconditions suitable for growth and, optionally, regeneration of atransgenic plant. In some embodiments, the transgenic plant is crossedwith a second plant of the same species, e.g., soybean, sunflower,canola or alfalfa.

In another aspect, the invention relates to methods of identifyingcandidate QTL associated with Sclerotinia stem rot resistance fromplants, particularly dicots. Such methods involve providing an isolatedor recombinant nucleic acid corresponding to a QTL as described above,and identifying a homolog of the nucleic acid in a plant, such as adicot. In one embodiment, the isolated or recombinant nucleic acids areprovided, and the homolog is identified in silico. In another embodimentthe isolated or recombinant nucleic acids are provided, and the homologis identified by nucleic acid hybridization under selectivehybridization conditions. In some embodiments, the homolog is isolated,e.g., cloned.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a QTL likelihood map for Sclerotinia stem rot resistance onsoybean linkage group A1. Molecular markers are placed on the left sidebar, and a Likelihood Ratio Statistic (LRS) is placed on the right sidebar. The number on the upper corner indicates the relative position ofthe LRS value generated by the MapManager program. The LRS peak of thisQTL is in the interval of Satt155-SLSIC.L24 with a distance of 1.3centiMorgans (cM).

FIG. 2 shows a QTL likelihood map for Sclerotinia stem rot resistance onsoybean linkage group A2. Molecular markers are placed on the left sidebar, and a Likelihood Ratio Statistic (LRS) is placed on the right sidebar. The number on the upper corner indicates the relative position ofthe LRS value generated by the MapManager program. The LRS peak of thisQTL is in the interval Sat_(—)129-Satt329 with a distance of 31.7 cM.

FIG. 3 shows a QTL likelihood map for Sclerotinia stem rot resistance onsoybean linkage group B2. Molecular markers are placed on the left sidebar, and a Likelihood Ratio Statistic (LRS) is placed on the right sidebar. The number on the upper corner indicates the relative position ofthe LRS value generated by the MapManager program. The LRS peak of thisQTL is in the interval Satt556-P1694 with a distance of 12.7 cM.

FIG. 4 shows a QTL likelihood map for Sclerotinia stem rot resistance onsoybean linkage group D2. Molecular markers are placed on the left sidebar, and a Likelihood Ratio Statistic (LRS) is placed on the right sidebar. The number on the upper corner indicates the relative position ofthe LRS value generated by the MapManager program. The LRS peak of thisQTL is in the interval PHP8701R-Satt311 with a distance of 14.5 cM.

FIG. 5 shows a QTL likelihood map for Sclerotinia stem rot resistance onsoybean linkage group E. Molecular markers are placed on the left sidebar, and a Likelihood Ratio Statistic (LRS) is placed on the right sidebar. The number on the upper corner indicates the relative position ofthe LRS value generated by the MapManager program. The LRS peak of thisQTL is in the interval PHP10118C-Satt231 with a distance of 18.8 cM.

FIG. 6 shows a QTL likelihood map for Sclerotinia stern rot resistanceon soybean linkage group J. Molecular markers are placed on the leftside bar, and a Likelihood Ratio Statistic (LRS) is placed on the rightside bar. The number on the upper corner indicates the relative positionof the LRS value generated by the MapManager program. The LRS peak ofthis QTL is in the interval P1047-A724_(—)1 with a distance of 22.5 cM.

FIG. 7 shows a QTL likelihood map for Sclerotinia stem rot resistance onsoybean linkage group L. Molecular markers are placed on the left sidebar, and a Likelihood Ratio Statistic (LRS) is placed on the right sidebar. The number on the upper corner indicates the relative position ofthe LRS value generated by the MapManager program. The LRS peak of thisQTL is in the interval Satt523-SLS2C.F20 with a distance of 11.7 cM.

DETAILED DISCUSSION Overview

The present invention relates to the identification of genetic markers,e.g., marker loci and nucleic acids corresponding to (or derived from)these marker loci, such as probes and amplification products useful forgenotyping plants, correlated with Sclerotinia stem rot resistance. Themarkers of the invention are used to identify plants, particularlyplants of the species Glycine max (soybean), that are resistant orexhibit improved resistance to Sclerotinia stem rot, or white mold.Accordingly, these markers are useful for marker-assisted selection andbreeding of Sclerotinia stem rot resistant plants, and foridentification of susceptible plants. The markers of the invention arealso used to identify and define chromosome intervals corresponding to,or including, quantitative trait loci associated with Sclerotinia stemrot resistance. Quantitative Trait Loci (QTL) associated withSclerotinia stem rot resistance are isolated by positional cloning,e.g., of genetic intervals defined by a pair of markers describedherein, or subsequences of an interval defined by and including suchmarkers. Such isolated QTL nucleic acids can be used for the productionof transgenic cells and plants exhibiting improved resistance toSclerotinia. In addition, QTL nucleic acids isolated from one organism,e.g., soybean, can, in turn, serve to isolate homologs of QTLs forSclerotinia stem rot resistance from other susceptible organisms,including a variety of commercially important dicots, such as canola,alfalfa, and sunflower.

DEFINITIONS

Units, prefixes, and symbols are denoted in their International Systemof Units (SI) accepted form. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; and amino acidsequences are written left to right in amino to carboxy orientation.Numeric ranges recited within the specification are inclusive of thenumbers defining the range and include each integer within the definedrange. Nucleotides may be referred to herein by their one-letter symbolsrecommended by the IUPAC-IUBMB Nomenclature Commission. The termsdefined below are more fully defined by reference to the specificationas a whole. Section headings provided throughout the specification areprovided for convenience and are not limitations to the various objectsand embodiments of the present invention.

The term “quantitative trait locus” or “QTL” refers to a polymorphicgenetic locus with at least two alleles that differentially affect theexpression of a continuously distributed phenotypic trait.

The term “associated with” or “associated” in the context of thisinvention refers to, e.g., a nucleic acid and a phenotypic trait, thatare in linkage disequilibrium, i.e., the nucleic acid and the trait arefound together in progeny plants more often than if the nucleic acid andphenotype segregated separately.

The term “linkage disequilibrium” refers to a non-random segregation ofgenetic loci. This implies that such loci are in sufficient physicalproximity along a length of a chromosome that they tend to segregatetogether with greater than random frequency.

The term “genetically linked” refers to genetic loci that are in linkagedisequilibrium and statistically determined not to assort independently.Genetically linked loci assort dependently from 51% to 99% of the timeor any whole number value therebetween, preferably at least 60%, 70%,80%, 90%, 95% or 99%.

The term “proximal” means genetically linked, typically within about 20centiMorgans (cM).

The term “marker” or “molecular marker” refers to a genetic locus (a“marker locus”) used as a point of reference when identifyinggenetically linked loci such as a QTL. The term also refers to nucleicacid sequences complementary to the genomic sequences, such as nucleicacids used as probes.

The term “interval” refers to a continuous linear span of chromosomalDNA with termini defined by and including molecular markers.

The terms “nucleic acid,” “polynucleotide,” “polynucleotide sequence”and “nucleic acid sequence” refer to single-stranded or double-strandeddeoxyribonucleotide or ribonucleotide polymers, or chimeras thereof. Asused herein, the term can additionally or alternatively include analogsof naturally occurring nucleotides having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides (e.g.,peptide nucleic acids). Unless otherwise indicated, a particular nucleicacid sequence of this invention optionally encompasses complementarysequences, in addition to the sequence explicitly indicated. The term“gene” is used to refer to, e.g., a cDNA and an mRNA encoded by thegenomic sequence, as well as to that genomic sequence.

The term “homologous” refers to nucleic acid sequences that are derivedfrom a common ancestral gene through natural or artificial processes(e.g., are members of the same gene family), and thus, typically, sharesequence similarity. Typically, homologous nucleic acids have sufficientsequence identity that one of the sequences or its complement is able toselectively hybridize to the other under selective hybridizationconditions. The term “selectively hybridizes” includes reference tohybridization, under stringent hybridization conditions, of a nucleicacid sequence to a specified nucleic acid target sequence to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences have about at least 80% sequence identity,preferably at least 90% sequence identity, and most preferably 95%, 97%,99%, or 100% sequence identity with each other. A nucleic acid thatexhibits at least some degree of homology to a reference nucleic acidcan be unique or identical to the reference nucleic acid or itscomplementary sequence.

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is substantially free from components that normallyaccompany or interact with it in its naturally occurring environment.The isolated material optionally comprises material not found with thematerial in its natural environment, e.g., a cell. In addition, if thematerial is in its natural environment, such as a cell, the material hasbeen placed at a location in the cell (e.g., genome or subcellularorganelle) not native to a material found in that environment. Forexample, a naturally occurring nucleic acid (e.g., a promoter) isconsidered to be isolated if it is introduced by non-naturally occurringmeans to a locus of the genome not native to that nucleic acid. Nucleicacids which are “isolated” as defined herein, are also referred to as“heterologous” nucleic acids.

The term “recombinant” indicates that the material (e.g., a nucleic acidor protein) has been synthetically (non-naturally) altered by humanintervention. The alteration to yield the synthetic material can beperformed on the material within or removed from its natural environmentor state. For example, a naturally occurring nucleic acid is considereda recombinant nucleic acid if it is altered, or if it is transcribedfrom DNA which has been altered, by means of human interventionperformed within the cell from which it originates. See, e.g., Compoundsand Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec,U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting inEukaryotic Cells; Zarling et al., WO119931022443.

The term “introduced” when referring to a heterologous or isolatednucleic acid refers to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid can beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). The term includes suchnucleic acid introduction means as “transfection,” “transformation” and“transduction.”

The term “host cell” means a cell which contains a heterologous nucleicacid, such as a vector, and supports the replication and/or expressionof the nucleic acid. Host cells may be prokaryotic cells such as E.coli, or eukaryotic cells such as yeast, insect, amphibian, or mammaliancells. Preferably, host cells are monocotyledonous or dicotyledonousplant cells. In the context of the invention, one particularly preferredmonocotyledonous host cell is a soybean host cell.

The term “transgenic plant” refers to a plant which comprises within itsgenome a heterologous polynucleotide. Generally, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant expression cassette. “Transgenic” is used herein to refer toany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenic organisms or cells initially so altered, aswell as those created by crosses or asexual propagation from the initialtransgenic organism or cell. The term “transgenic” as used herein doesnot encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods (i.e.,crosses) or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

The term “dicot” refers to the subclass of angiosperm plants also knowsas “dicotyledoneae” and includes reference to whole plants, plant organs(e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny ofthe same. Plant cell, as used herein includes, without limitation,seeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores.

The term “crossed” or “cross” in the context of this invention means thefusion of gametes via pollination to produce progeny (i.e., cells,seeds, or plants). The term encompasses both sexual crosses (thepollination of one plant by another) and selfing (self-pollination,i.e., when the pollen and ovule are from the same plant).

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny plant via a sexual cross between twoparent plants, where at least one of the parent plants has the desiredallele within its genome. Alternatively, for example, transmission of anallele can occur by recombination between two donor genomes, e.g., in afused protoplast, where at least one of the donor protoplasts has thedesired allele in its genome. The desired allele can be, e.g., atransgene or a selected allele of a marker or QTL.

Markers

The present invention provides molecular markers genetically linked toquantitative trait loci (“QTL”) associated with resistance toSclerotinia stem rot, also known as white mold. Such molecular markersare useful for identifying and producing dicotyledonous plants, inparticular, such commercially important dicot crops as sunflower,canola, alfalfa, and soybean, resistant, or with improved resistance, toSclerotinia stem rot.

Genetic mapping of over 300 molecular markers has developed a geneticlinkage map covering approximately 2400 cM (centiMorgans) correspondingto the 20 soybean chromosomes. Of 331 markers mapped, 53 are restrictionfragment length polymorphisms (RFLP), 159 are amplified fragment lengthpolymorphisms (AFLP), 21 are proprietary (Dupont) simple sequence repeat(SSR) polymorphisms, and 100 are public SSR polymorphisms. Additionaldetails regarding the nature and use of molecular markers are providedbelow in the section entitled “MARKER ASSISTED SELECTION AND BREEDING.”

Exemplary marker loci associated with resistance to Sclerotinia stem rotare localized to seven linkage groups in soybean: A1, A2, B2, D2, E, J,and L. These exemplary marker loci delineate chromosomal intervalsincluding Quantitative Trait Loci (QTL) associated with phenotypicmeasures of resistance to Sclerotinia stem rot. For example: Satt155 andSLS1C.L24 localize to linkage group A1; Sat_(—)129 and Satt329 localizeto linkage group A2; Satt556 and P1694 localize to linkage group B2;PHP8701R and Satt311 localize to linkage group D2; PHP10118C and Satt231localize to linkage group E; P1047 and A724_(—)1 localize to linkagegroup 3; and Satt523 and SLS2C.F20 localize to linkage group L.

Simple sequence repeat (SSR) markers P1694, Sat_(—)129, Satt155,Satt231, P1047, Satt311, Satt329, PHP10118C, Satt523, and Satt556 arediscussed in Cregan et al. (1994) Meth. Mol. Cell. Biol. 5:49-61, areavailable from the SoyBase internet database established by the UnitedStates Department of Agriculture (SoyBase, G304 Agronomy Hall Iowa StateUniversity, Ames, Iowa). Primers suitable for amplification ofpolymorphic products corresponding to these marker loci are provided inTable 1. Additional primers and probes corresponding to these markerscan be designed based on the sequence information provided hereinbelow(SEQ ID NO:24-33). In addition, substitute primers and probes formarkers P1694, PHP10118C and P1047 can be selected based on thesequences of the linked publicly available markers Satt063, Sat_(—)124,and Satt596, respectively.

Marker A724_(—)1 is detectable as an RFLP using a publicly availableprobe (SoyBase, G304 Agronomy Hall Iowa State University, Ames, Iowa;internet address: http://129.186.26.94).

SLS1C.L24 (SEQ ID NO:1) and SLS2C.F20 (SEQ ID NO:2) are novel expressedsequence tag (EST) markers used as probes for detecting restrictionfragment length polymorphisms, e.g., by Southern analysis.

PHP8701R (SEQ ID NO:3) is a novel marker locus detected as an amplifiedfragment length polymorphism.

TABLE 1 Primers for amplification of Marker Loci Marker Forward PrimerReverse Primer P1694 TGTCAATAAATGGCTAATCG TGGTAGTTTTCACACGTTATG(SEQ ID NO: 4) (SEQ ID NO: 5) Sat_129 TTCAGTACAAGTCGGGTGAATAATAATATCACATGTTCGGGACTTAAGGTAT (SEQ ID NO: 6) (SEQ ID NO: 7) Satt155AGATCCAACACCTGGCCTAAT GCTGCACAATTCATTCCATTT (SEQ ID NO: 8)(SEQ ID NO: 9) Satt231 GCGTGTGCAAAATGTTCATCATCT GGCACGAATCAACATCAAAACTTC(SEQ ID NO: 10) (SEQ ID NO: 11) P1047 TCGCTTTTAATGACCTCGACCGGAGGATTTGTCATAGATTC (SEQ ID NO: 12) (SEQ ID NO: 13) Satt311GGGGGAACCACAAAAATCTTAATC GTTGAAGCTCAGGCTGTGATGAAT (SEQ ID NO: 14)(SEQ ID NO: 15) Satt329 GCGGGACGCAAAATTGGATTTAGTGCGCCGAATAAAACGTGAGAACTG (SEQ ID NO: 16) (SEQ ID NO: 17) PHP10118CGACTGCGTACCAATTCACA GATGAGTCCTGAGTAACAC (SEQ ID NO: 18) (SEQ ID NO: 19)Satt523 GCGATTTCTTCCTTGAAGAATTTTCTG GCGCTTTTTCGGCTGTTATTTTTAACT(SEQ ID NO: 20) (SEQ ID NO: 21) Satt556 GCGATAAAACCCGATAAATAAGCGTTCTGCACCTTGTTTTCT (SEQ ID NO: 22) (SEQ ID NO: 23)

It will be noted that, regardless of their molecular nature, e.g.,whether the marker is an SSR, AFLP, RFLP, etc., markers are typicallystrain specific. That is, a particular polymorphic marker, such as theexemplary markers of the invention described above, is defined relativeto the parental lines of interest. For each marker locus,resistance-associated, and conversely, susceptibility-associated allelesare identified for each pair of parental lines. Following correlation ofspecific alleles with susceptibility and resistance in parents of across, the marker can be utilized to identify progeny with genotypesthat correspond to the desired resistance phenotype. In somecircumstance, i.e., in some crosses of parental lines, the exemplarymarkers described herein will not be optimally informative. In suchcases, additional informative markers, e.g., certain linked markersand/or homologous markers are evaluated and substituted for genotyping,e.g., for marker-assisted selection, etc. In the case where a markercorresponds to a QTL, following identification of resistance- andsusceptibility-associated alleles, it is possible to directly screen apopulation of samples, e.g., samples obtained from a seed bank, withoutfirst correlating the parental phenotype with an allele.

Linked Markers

FIGS. 1-7 provide additional linked markers that can be used in additionto, or in place of: Satt155, SLS1C.L24, Sat_(—)129, Satt329, Satt556,P1694, PHP8701R, Satt311, PHP10118C, Satt231, P1047, A724_(—)1, Satt523,and SLS2C.F20 for the purposes, e.g., of mapping and isolating QTLassociated with Sclerotinia stem rot. Those of skill in the art willrecognize that additional molecular markers can be identified within theintervals defined by the above-described pairs of markers. Such markersare also genetically linked to the QTLs identified herein as associatedwith Sclerotinia rot resistance, and are within the scope of the presentinvention. Markers can be identified by any of a variety of genetic orphysical mapping techniques. Methods of determining whether markers aregenetically linked to a QTL (or to a specified marker) associated withresistance to Sclerotinia stem rot are known to those of skill in theart and include, e.g., interval mapping (Lander and Botstein (1989)Genetics 121:185), regression mapping (Haley and Knott (1992) Heredity69:315) or MQM mapping (Jansen (1994) Genetics 138:871). In addition,such physical mapping techniques as chromosome walking, contig mappingand assembly, and the like, can be employed to identify and isolateadditional sequences useful as markers in the context of the presentinvention.

Homologous Markers

In addition, Satt155, SLS1C.L24, Sat_(—)129, Satt329, Satt556, P1694,PHP8701R, Satt311, PHP10118C, Satt231, P1047, A724_(—)1, Satt523, andSLS2C.F20 are useful for the identification of homologous markersequences with utility in identifying QTL associated with Sclerotiniastem rot resistance in different lines, varieties, or species of dicots.Such homologous markers are also a feature of the invention.

Homologous markers can be identified by selective hybridization to areference sequence. The reference sequence is typically a uniquesequence, such as unique oligonucleotide primer sequences, ESTs,amplified fragments (e.g., corresponding to AFLP markers) and the like,derived from the marker loci Satt155, SLSIC.L24, Sat_(—)129, Satt329,Satt556, P1694, PHP8701R, Satt311, PHP10118C, Satt231, P1047, A724_(—)1,Satt523, and SLS2C.F20, or its complement.

Two single-stranded nucleic acids “hybridize” when they form adouble-stranded duplex. The double stranded region can include thefull-length of one or both of the single-stranded nucleic acids, or allof one single stranded nucleic acid and a subsequence of the othersingle-stranded nucleic acid, or the double stranded region can includea subsequence of each nucleic acid. Selective hybridization conditionsdistinguish between nucleic acids that are related, e.g., sharesignificant sequence identity with the reference sequence (or itscomplement) and those that associate with the reference sequence in anon-specific manner Generally, selective hybridization conditions arethose in which the salt concentration is less than about 1.5 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Selective hybridizationconditions may also be achieved with the addition of destabilizingagents such as formamide. Selectivity can be achieved by varying thestringency of the hybridization and/or wash conditions. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically a function of post-hybridization washes, withthe critical factors being ionic strength and temperature of the finalwash solution. Generally, stringent conditions are selected to be about5° C. lower than the thermal melting point (T_(m)) for the specificsequence and its complement at a defined ionic strength and pH. However,severely stringent conditions can utilize a hybridization and/or wash at1, 2, 3, or 4° C. lower than the thermal melting point (T_(m));moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T_(m));low stringency conditions can utilize a hybridization and/or wash at 11,12, 13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of a complementary target sequence hybridizes to a perfectlymatched probe. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)-500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with ≧90% identity are sought, the T_(m) can be decreased10° C.

Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution) it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. Hybridization and/or wash conditions can be applied for atleast 10, 30, 60, 90, 120, or 240 minutes. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes Part I, Chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,N.Y. General Texts which discuss considerations relevant to nucleic acidhybridization, the selection of probes, and buffer and incubationconditions, and the like, as well as numerous other topics of interestin the context of the present invention (e.g., cloning of nucleic acidswhich correspond to markers and QTL, sequencing of cloned markers/QTL,the use of promoters, vectors, etc.) can be found in Berger and Kimmel(1987) Guide to Molecular Cloning Techniques, Methods in Enzymology vol.152, Academic Press, Inc., San Diego (“Berger”); Sambrook et al.,(2001)Molecular Cloning—A Laboratory Manual, 3^(rd) ed. Vols. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor (“Sambrook”); and Ausubelet al., (eds) (supplemented through 2001)Current Protocols in MolecularBiology, John Wiley and Sons, Inc., (“Ausubel”).

In addition to hybridization methods described above, homologs of themarkers of the invention can be identified in silico using any of avariety of sequence alignment and comparison protocols. For the purposesof the ensuing discussion, the following terms are used to describe thesequence relationships between a marker nucleotide sequence and areference polynucleotide sequence:

A “reference sequence” is a defined sequence used as a basis forsequence comparison with a test sequence, e.g., a candidate markerhomolog, of the present invention. A reference sequence may be asubsequence or the entirety of a specified sequence; for example, asegment of a full-length cDNA or gene sequence, or the complete cDNA orgene sequence.

As used herein, a “comparison window” is a contiguous and specifiedsegment, (e.g., a subsequence) of a polynucleotide/polypeptide sequenceto be compared to a reference sequence. The segment of thepolynucleotide/polypeptide sequence in the comparison window can includeone or more additions or deletions (i.e., gaps) with respect to thereference sequence, which (by definition) does not comprise addition(s)or deletion(s), for optimal alignment of the two sequences. An optimalalignment of two sequences yields the fewest number of unlikenucleotide/amino acid residues in a comparison window. Generally, thecomparison window is at least 20 contiguous nucleotide/amino acidresidues in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a falsely highsimilarity between two sequences, due to inclusion of gaps in thepolynucleotide/polypeptide sequence, a gap penalty is typically assessedand is subtracted from the number of matches.

“Sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences refers to residues that are the same in bothsequences when aligned for maximum correspondence over a specifiedcomparison window.

“Percentage sequence identity” refers to the value determined bycomparing two optimally aligned sequences over a comparison window. Thepercentage is calculated by determining the number of positions at whichboth sequences have the same nucleotide or amino acid residue,determining the number of matched positions, dividing the number ofmatched positions by the total number of positions in the comparisonwindow, and multiplying the result by 100 to yield the percentage ofsequence identity.

When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer by conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller (1988) Computer Applic. Biol. Sci.4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482; by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443; by the search for similarity method ofPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp (1988) Gene 73:237-244; Higgins and Sharp (1989)CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Research16:10881-90; Huang et al. (1992) Computer Applications in theBiosciences 8: 155-65, and Pearson et al. (1994) Methods in MolecularBiology 24:307-331.

The BLAST family of programs which can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, e.g., CurrentProtocols in Molecular Biology, Chapter 19, Ausubel et al., Eds., (1995)Greene Publishing and Wiley-Interscience, New York; Altschul et al.(1990) J. Mol. Biol. 215:403-410; and, Altschul et al. (1997) NucleicAcids Res. 25:3389-3402.

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information (available onthe internet). This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold. These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see, e.g., Henikoff & Henikoff (1989) Proc.Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5877). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences.However, many real proteins comprise regions of nonrandom sequenceswhich may be homopolymeric tracts, short-period repeats, or regionsenriched in one or more amino acids. Such low-complexity regions may bealigned between unrelated proteins even though other regions of theprotein are entirely dissimilar. A number of low-complexity filterprograms can be employed to reduce such low-complexity alignments. Forexample, the SEG (Wooten and Federhen (1993) Comput. Chem. 17:149-163)and XNU (Claverie and States (1993) Comput. Chem. 17:191-201)low-complexity filters can be employed alone or in combination.

Unless otherwise stated, nucleotide and protein identity/similarityvalues provided herein are calculated using GAP (GCG Version 10) underdefault values.

GAP (Global Alignment Program) can also be used to compare apolynucleotide or polypeptide of the present invention with a referencesequence. GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48: 443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 100. Thus, for example, the gapcreation and gap extension penalties can each independently be: 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see, e.g., Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

Multiple alignment of the sequences can be performed using the CLUSTALmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the CLUSTAL method are KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

The percentage sequence identity of a homologous marker to its referencemarker (e.g., any one Satt155, SLS1C.L24, Sat_(—)129, Satt329, Satt556,P1694, PHP8701R, Satt311, PHP10118C, Satt231, P1047, A724_(—)1, Satt523,and SLS2C.F20) is typically at least 80% and, rounded upwards to thenearest integer, can be expressed as an integer selected from the groupof integers between 80 and 99. Thus, for example, the percentagesequence identity to a reference sequence can be at least 80%, 85%, 90%,95%, 97%, or 99%. Sequence identity can be calculated using, forexample, the BLAST, CLUSTALW, or GAP algorithms under defaultconditions.

Detection of Marker Loci

Markers corresponding to genetic polymorphisms between members of apopulation can be detected by numerous methods, well-established in theart (e.g., restriction fragment length polymorphisms, isozyme markers,allele specific hybridization (ASH), amplified variable sequences of theplant genome, self-sustained sequence replication, simple sequencerepeat (SSR), single nucleotide polymorphism (SNP), or amplifiedfragment length polymorphisms (AFLP)).

The majority of genetic markers rely on one or more property of nucleicacids for their detection. For example, some techniques for detectinggenetic markers utilize hybridization of a probe nucleic acid to nucleicacids corresponding to the genetic marker. Hybridization formatsincluding but not limited to, solution phase, solid phase, mixed phase,or in situ hybridization assays. Markers which are restriction fragmentlength polymorphisms (RFLP), are detected by hybridizing a probe whichis typically a sub-fragment (or a synthetic oligonucleotidecorresponding to a sub-fragment) of the nucleic acid to be detected torestriction digested genomic DNA. The restriction enzyme is selected toprovide restriction fragments of at least two alternative (orpolymorphic) lengths in different individuals, and will often vary fromline to line. Determining a (one or more) restriction enzyme thatproduces informative fragments for each cross is a simple procedure,well known in the art. After separation by length in an appropriatematrix (e.g., agarose) and transfer to a membrane (e.g., nitrocellulose,nylon), the labeled probe is hybridized under conditions which result inequilibrium binding of the probe to the target followed by removal ofexcess probe by washing.

Nucleic acid probes to the marker loci can be cloned and/or synthesized.Detectable labels suitable for use with nucleic acid probes include anycomposition detectable by spectroscopic, radioisotopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels include biotin for staining with labeled streptavidinconjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes, andcolorimetric labels. Other labels include ligands which bind toantibodies labeled with fluorophores, chemiluminescent agents, andenzymes. Labeling markers is readily achieved such as by the use oflabeled PCR primers to marker loci.

The hybridized probe is then detected using, most typically byautoradiography or other similar detection technique (e.g.,fluorography, liquid scintillation counter, etc.). Examples of specifichybridization protocols are widely available in the art, see, e.g.,Berger, Sambrook, Ausubel, all supra.

Amplified variable sequences refer to amplified sequences of the plantgenome which exhibit high nucleic acid residue variability betweenmembers of the same species. All organisms have variable genomicsequences and each organism (with the exception of a clone) has adifferent set of variable sequences. Once identified, the presence ofspecific variable sequence can be used to predict phenotypic traits.Preferably, DNA from the plant serves as a template for amplificationwith primers that flank a variable sequence of DNA. The variablesequence is amplified and then sequenced.

In vitro amplification techniques are well known in the art. Examples oftechniques sufficient to direct persons of skill through such in vitromethods, including the polymerase chain reaction (PCR), the ligase chainreaction (LCR), Qβ-replicase amplification and other RNA polymerasemediated techniques (e.g., NASBA), are found in Berger, Sambrook andAusubel (all supra) as well as Mullis et al. (1987) U.S. Pat. No.4,683,202; PCR Protocols, A Guide to Methods and Applications (Innis etal., eds.) Academic Press Inc., San Diego Academic Press Inc. San Diego,Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; TheJournal Of NIH Research (1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl.Acad. Sci. USA 86, 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci.USA 87, 1874; Lomell et al. (1989) J. Clin. Chem. 35, 1826; Landegren etal., (1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8,291-294; Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990)Gene 89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564.Improved methods of cloning in vitro amplified nucleic acids aredescribed in Wallace et al., U.S. Pat. No. 5,426,039. Improved methodsof amplifying large nucleic acids by PCR are summarized in Cheng et al.(1994) Nature 369: 684, and the references therein, in which PCRamplicons of up to 40 kb are generated. One of skill will appreciatethat essentially any RNA can be converted into a double stranded DNAsuitable for restriction digestion, PCR expansion and sequencing usingreverse transcriptase and a polymerase. See, Ausubel, Sambrook andBerger, all supra.

Oligonucleotides for use as primers, e.g., in amplification reactionsand for use as nucleic acid sequence probes are typically synthesizedchemically according to the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers (1981) Tetrahedron Lett. 22:1859, orcan simply be ordered commercially.

Alternatively, self-sustained sequence replication can be used toidentify genetic markers. Self-sustained sequence replication refers toa method of nucleic acid amplification using target nucleic acidsequences which are replicated exponentially in vitro undersubstantially isothermal conditions by using three enzymatic activitiesinvolved in retroviral replication: (1) reverse transcriptase, (2) RnaseH, and (3) a DNA-dependent RNA polymerase (Guatelli et al. (1990) ProcNatl Acad Sci USA 87:1874). By mimicking the retroviral strategy of RNAreplication by means of cDNA intermediates, this reaction accumulatescDNA and RNA copies of the original target.

Amplified fragment length polymorphisms (AFLP) can also be used asgenetic markers (Vos et al. (1995) Nucl Acids Res 23:4407. The phrase“amplified fragment length polymorphism” refers to selected restrictionfragments which are amplified before or after cleavage by a restrictionendonuclease. The amplification step allows easier detection of specificrestriction fragments. AFLP allows the detection large numbers ofpolymorphic markers and has been used for genetic mapping of plants(Becker et al. (1995) Mol Gen Genet. 249:65; and Meksem et al. (1995)Mol Gen Genet. 249:74.

Allele-specific hybridization (ASH) can be used to identify the geneticmarkers of the invention. ASH technology is based on the stableannealing of a short, single-stranded, oligonucleotide probe to acompletely complementary single-strand target nucleic acid. Detection isvia an isotopic or non-isotopic label attached to the probe.

For each polymorphism, two or more different ASH probes are designed tohave identical DNA sequences except at the polymorphic nucleotides. Eachprobe will have exact homology with one allele sequence so that therange of probes can distinguish all the known alternative allelesequences. Each probe is hybridized to the target DNA. With appropriateprobe design and hybridization conditions, a single-base mismatchbetween the probe and target DNA will prevent hybridization. In thismanner, only one of the alternative probes will hybridize to a targetsample that is homozygous or homogenous for an allele. Samples that areheterozygous or heterogeneous for two alleles will hybridize to both oftwo alternative probes.

ASH markers are used as dominant markers where the presence or absenceof only one allele is determined from hybridization or lack ofhybridization by only one probe. The alternative allele may be inferredfrom the lack of hybridization. ASH probe and target molecules areoptionally RNA or DNA; the target molecules are any length ofnucleotides beyond the sequence that is complementary to the probe; theprobe is designed to hybridize with either strand of a DNA target; theprobe ranges in size to conform to variously stringent hybridizationconditions, etc.

PCR allows the target sequence for ASH to be amplified from lowconcentrations of nucleic acid in relatively small volumes. Otherwise,the target sequence from genomic DNA is digested with a restrictionendonuclease and size separated by gel electrophoresis. Hybridizationstypically occur with the target sequence bound to the surface of amembrane or, as described in U.S. Pat. No. 5,468,613, the ASH probesequence may be bound to a membrane.

In one embodiment, ASH data are obtained by amplifying nucleic acidfragments (amplicons) from genomic DNA using PCR, transferring theamplicon target DNA to a membrane in a dot-blot format, hybridizing alabeled oligonucleotide probe to the amplicon target, and observing thehybridization dots by autoradiography.

Single nucleotide polymorphisms (SNP) are markers that consist of ashared sequence differentiated on the basis of a single nucleotide.Typically, this distinction is detected by differential migrationpatterns of an amplicon comprising the SNP on e.g., an acrylamide gel.However, alternative modes of detection, such as hybridization, e.g.,ASH, or RFLP analysis are not excluded.

In yet another basis for providing a genetic linkage map, Simplesequence repeats (SSR), take advantage of high levels of di-, tri-, ortetra-nucleotide tandem repeats within a genome. Dinucleotide repeatshave been reported to occur in the human genome as many as 50,000 timeswith n varying from 10 to 60 or more (Jacob et al. (1991) Cell 67:213.Dinucleotide repeats have also been found in higher plants (Condit andHubbell (1991) Genome 34:66).

Briefly, SSR data is generated by hybridizing primers to conservedregions of the plant genome which flank the SSR sequence. PCR is thenused to amplify the dinucleotide repeats between the primers. Theamplified sequences are then electorphoresed to determine the size andtherefore the number of di-, tri-, and tetra-nucleotide repeats.

Alternatively, isozyme markers are employed as genetic markers. Isozymesare multiple forms of enzymes which differ from one another in theiramino acid, and therefore their nucleic acid sequences. Some isozymesare multimeric enzymes containing slightly different subunits. Otherisozymes are either multimeric or monomeric but have been cleaved fromthe proenzyme at different sites in the amino acid sequence. Isozymescan be characterized and analyzed at the protein level, oralternatively, isozymes which differ at the nucleic acid level can bedetermined. In such cases any of the nucleic acid based methodsdescribed herein can be used to analyze isozyme markers.

In alternative embodiments, in silico methods can be used to detect themarker loci. For example, the sequence of a nucleic acid comprising themarker can be stored in a computer. The desired marker locus sequence orits homolog can be identified using an appropriate nucleic acid searchalgorithm as provided by, for example, in such readily availableprograms as BLAST.

QTL Mapping

Multiple experimental paradigms have been developed to identify andanalyze QTL. In general, these paradigms involve crossing one or moreparental pairs, which can be, for example, a single pair derived fromtwo inbred strains, or multiple related or unrelated parents ofdifferent inbred strains or lines, which each exhibit differentcharacteristics relative to the phenotypic trait of interest. Theparents, and a population of progeny are genotyped, typically formultiple marker loci, and evaluated for the trait of interest. In thecontext of the present invention, the parental and progeny plants aregenotyped for any one or more of the molecular markers: Satt155,SLS1C.L24, Sat_(—)129, Satt329, Satt556, P1694, PHP8701R, Satt311,PHP10118C, Satt231, P1047, A724_(—)1, Satt523, and SLS2C.F20, orhomologues, or alternative markers linked to any one or more of Satt155,SLS1C.L24, Sat_(—)129, Satt329, Satt556, P1694, PHP8701R, Satt311,PHP10118C, Satt231, P1047, A724_(—)1, Satt523, and SLS2C.F20, andevaluated for resistance, or relative resistance to Sclerotinia stemrot. QTL associated with Sclerotinia stem rot resistance are identifiedbased on the significant statistical correlations between the markergenotype(s) and the resistance phenotype of the evaluated progenyplants. Numerous methods for determining whether markers are geneticallylinked to a QTL (or to another marker) associated with resistance toSclerotinia stem rot are known to those of skill in the art and include,e.g., interval mapping (Lander and Botstein (1989) Genetics 121:185),regression mapping (Haley and Knott (1992) Heredity 69:315) or MQMmapping (Jansen (1994) Genetics 138:871). In addition, the followingapplications provide additional details regarding alternativestatistical methods applicable to complex breeding populations which canbe used to identify and localize QTLs associated with Sclerotinia stemrot resistance: U.S. Pat. No. 6,399,855, issued Jun. 4, 2002, by Beaviset al. “QTL MAPPING IN PLANT BREEDING POPULATIONS” and WO 2001/049104,published Jul. 12, 2001, by Jansen et al. “MQM MAPPING USING HAPLOTYPEDPUTATIVE QTLS ALLELES: A SIMPLE APPROACH FOR MAPPING QTLS IN PLANTBREEDING POPULATIONS.”

Marker Assisted Selection and Breeding of Plants

A primary motivation for development of molecular markers in cropspecies is the potential for increased efficiency in plant breedingthrough marker assisted selection (MAS). Genetic marker alleles, oralternatively, identified QTL alleles, are used to identify plants thatcontain a desired genotype at one or more loci, and that are expected totransfer the desired genotype, along with a desired phenotype to theirprogeny. Genetic marker alleles (or QTL alleles) can be used to identifyplants that contain a desired genotype at one locus, or at severalunlinked or linked loci (e.g., a haplotype), and that would be expectedto transfer the desired genotype, along with a desired phenotype totheir progeny. The present invention provides the means to identifyplants, particularly dicots, e.g., soybean, that are resistant, orexhibit improved resistance, to Sclerotinia stem rot by identifyingplants having a specified allele, e.g., at markers Satt155, SLS1C.L24,Sat_(—)129, Satt329, Satt556, P1694, PHP8701R, Satt311, PHP10118C,Satt231, P1047, A724_(—)1, Satt523, and SLS2C.F20, or homologous orlinked markers. Similarly, by identifying plants lacking the desiredallele, susceptible plants can be identified, and, e.g., eliminated fromsubsequent crosses. It will be appreciated that for the purposes of MAS,the term marker can encompass both marker and QTL loci as both can beused to identify plants with that are resistant or have improvedresistance to Sclerotinia stem rot.

After a desired phenotype, e.g., Sclerotinia stem rot resistance, and apolymorphic chromosomal locus, e.g., a marker locus or QTL, aredetermined to segregate together, it is possible to use thosepolymorphic loci to select for alleles corresponding to the desiredphenotype—a process called marker-assisted selection (MAS). In brief, anucleic acid corresponding to the marker nucleic acid is detected in abiological sample from a plant to be selected. This detection can takethe from of hybridization of a probe nucleic acid to a marker, e.g.,using allele-specific hybridization, Southern analysis, northernanalysis, in situ hybridization, hybridization of primers followed byPCR amplification of a region of the marker or the like. A variety ofprocedures for detecting markers are described herein, e.g., in thesection entitled “DETECTION OF MARKER LOCI.” After the presence (orabsence) of a particular marker in the biological sample is verified,the plant is selected, i.e., used to make progeny plants by selectivebreeding.

Soybean breeders need to combine disease resistance loci with genes forhigh yield and other desirable traits to develop improved soybeanvarieties. Disease screening for large numbers of samples can beexpensive, time consuming, and unreliable. Use of the polymorphic locidescribed herein, and genetically-linked nucleic acids, as geneticmarkers for disease resistance loci is an effective method for selectingresistant varieties in breeding programs. For example, one advantage ormarker-assisted selection over field evaluations for disease resistanceis that MAS can be done at any time of year regardless of the growingseason. Moreover, environmental effects are irrelevant tomarker-assisted selection.

When a population is segregating for multiple loci affecting one ormultiple traits, e.g., multiple loci involved in resistance to singledisease, or multiple loci each involved in resistance to differentdiseases, the efficiency of MAS compared to phenotypic screening becomeseven greater because all the loci can be processed in the lab togetherfrom a single sample of DNA. In the present instance, this means thatmultiple markers selected from among Satt155, SLS1C.L24, Sat_(—)129,Satt329, Satt556, P1694, PHP8701R, Satt311, PHP10118C, Satt231, P1047,A724_(—)1, Satt523, and SLS2C.F20, or markers homologous or linkedthereto can be assayed simultaneously or sequentially in a single sampleor population of samples. Thus, any one or more of these markers, e.g.,two or more, up to and including all of the established markers, can beassayed simultaneously. In some instances, it is desirable to evaluate amarker corresponding to each of the linkage groups associated withSclerotinia stem rot.

Another use of MAS in plant breeding is to assist the recovery of therecurrent parent genotype by backcross breeding. Backcross breeding isthe process of crossing a progeny back to one of its parents.Backcrossing is usually done for the purpose of introgressing one or afew loci from a donor parent into an otherwise desirable geneticbackground from the recurrent parent. The more cycles of backcrossingthat is done, the greater the genetic contribution of the recurrentparent to the resulting variety. This is often necessary, becauseresistant plants may be otherwise undesirable, i.e., due to low yield,low fecundity, or the like. In contrast, strains which are the result ofintensive breeding programs may have excellent yield, fecundity or thelike, merely being deficient in one desired trait such as resistance toa particular pathogen (e.g., Sclerotinia stem rot resistance).

The presence and/or absence of a particular genetic marker allele, e.g.,Satt155, SLS1C.L24, Sat_(—)129, Satt329, Satt556, P1694, PHP8701R,Satt311, PHP10118C, Satt231, P1047, A724_(—)1, Satt523, and SLS2C.F20,or a homolog thereof, in the genome of a plant exhibiting a preferredphenotypic trait is made by any method listed above, e.g., RFLP, AFLP,SSR, etc. If the nucleic acids from the plant are positive for a desiredgenetic marker, the plant can be selfed to create a true breeding linewith the same genotype, or it can be crossed with a plant with the samemarker or with other desired characteristics to create a sexuallycrossed hybrid generation.

Positional Cloning

The molecular markers of the present invention, e.g., Satt155,SLS1C.L24, Sat_(—)129, Satt329, Satt556, P1694, PHP8701R, Satt311,PHP10118C, Satt231, P1047, A724_(—)1, Satt523, and SLS2C.F20, andnucleic acids homologous thereto, can be used, as indicated previously,to identify additional linked marker loci, which can be cloned by wellestablished procedures, e.g., as described in detail in Ausubel, Bergerand Sambrook, supra. Similarly, the markers: Satt155, SLS1C.L24,Sat_(—)129, Satt329, Satt556, P1694, PHP8701R, Satt311, PHP10118C,Satt231, P1047, A724_(—)1, Satt523, and SLS2C.F20, as well as anyadditionally identified linked molecular markers can be used tophysically isolate, e.g., by cloning, nucleic acids associated with QTLscontributing to Sclerotinia stem rot resistance. Such nucleic acids,i.e., linked to QTL, have a variety of uses, including as geneticmarkers for identification of additional QTLs in subsequent applicationsof marker assisted selection (MAS).

These nucleic acids are first identified by their genetic linkage tomarkers of the present invention. Isolation of the nucleic acid ofinterest is achieved by any number of methods as discussed in detail insuch references as Ausubel, Berger and Sambrook, supra, and Clark, Ed.(1997) Plant Molecular Biology: A Laboratory Manual Springer-Verlag,Berlin.

For example, “Positional gene cloning” uses the proximity of a geneticmarker to physically define an isolated chromosomal fragment that islinked to a QTL. The isolated chromosomal fragment can be produced bysuch well known methods as digesting chromosomal DNA with one or morerestriction enzymes, or by amplifying a chromosomal region in apolymerase chain reaction (PCR), or alternative amplification reaction.The digested or amplified fragment is typically ligated into a vectorsuitable for replication, e.g., a plasmid, a cosmid, a phage, anartificial chromosome, or the like, and, optionally expression, of theinserted fragment. Markers which are adjacent to an open reading frame(ORF) associated with a phenotypic trait can hybridize to a DNA clone,thereby identifying a clone on which an ORE is located. If the marker ismore distant, a fragment containing the open reading frame is identifiedby successive rounds of screening and isolation of clones which togethercomprise a contiguous sequence of DNA, a “contig.” Protocols sufficientto guide one of skill through the isolation of clones associated withlinked markers are found in, e.g. Berger, Sambrook and Ausubel, allsupra.

Isolated Chromosome Intervals

The present invention provides isolated nucleic acids comprising a QTLassociated with Sclerotinia stem rot resistance. The QTL is localizedwithin an interval defined by two markers of the present inventionwherein each marker flanks and is genetically linked to the QTL. Suchintervals can be utilized to identify homologous nucleic acids and/orcan be used in the production of transgenic plants having the resistanceto Sclerotinia stem rot conferred by the introduced QTL. A chromosomeinterval comprising a QTL is isolated, e.g., cloned via positionalcloning methods outlined above. A chromosome interval can contain one ormore ORFs associated with resistance, and can be cloned on one or moreindividual vectors, e.g., depending on the size of the chromosomeinterval.

It will be appreciated that numerous vectors are available in the artfor the isolation and replication of the nucleic acids of the invention.For example, plasmids, cosmids and phage vectors are well known in theart, and are sufficient for many applications (e.g., in applicationsinvolving insertion of nucleic acids ranging from less than 1 to about20 kilobases (kb). In certain applications, it is advantageous to makeor clone large nucleic acids to identify nucleic acids more distantlylinked to a given marker, or to isolate nucleic acids in excess of 10-20kb, e.g., up to several hundred kilobases or more, such as the entireinterval between two linked markers, i.e., up to and including one ormore centiMorgans (cM), linked to QTLs as identified herein. In suchcases, a number of vectors capable of accommodating large nucleic acidsare available in the art, these include, yeast artificial chromosomes(YACs), bacterial artificial chromosomes (BACs), plant artificialchromosomes (PACs) and the like. For a general introduction to YACs,BACs, PACs and MACs as artificial chromosomes, see, e.g., Monaco andLarin (1994) Trends Biotechnol 12:280. In addition, methods for the invitro amplification of large nucleic acids linked to genetic markers arewidely available (e.g., Cheng et al. (1994) Nature 369:684, andreferences therein). Cloning systems can be created or obtained fromcommercially; see, for example, Stratagene Cloning Systems, Catalogs2000 (La Jolla, Calif.).

Generation of Transgenic Plants and Cells

The present invention also relates to host cells and organisms which aretransformed with nucleic acids corresponding to QTL and other genesidentified according to the invention. For example, such nucleic acidsinclude chromosome intervals, ORFs, and/or cDNAs or corresponding to asequence or subsequence included within the identified chromosomeinterval or ORF. Additionally, the invention provides for the productionof polypeptides corresponding to QTL by recombinant techniques. Hostcells are genetically engineered (i.e., transduced, transfected ortransformed) with the vectors of this invention (i.e., vectors whichcomprise QTLs or other nucleic acids identified according to the methodsof the invention and as described above) which are, for example, acloning vector or an expression vector. Such vectors include, inaddition to those described above, e.g., an agrobacterium, a virus (suchas a plant virus), a naked polynucleotide, or a conjugatedpolynucleotide. The vectors are introduced into plant tissues, culturedplant cells or plant protoplasts by a variety of standard methodsincluding electroporation (From et al. (1985) Proc. Natl. Acad. Sci. USA82; 5824), infection by viral vectors such as cauliflower mosaic virus(CaMV) (Hohn et al. (1982) Molecular Biology of Plant Tumors (AcademicPress, New York, pp. 549-560; Howell U.S. Pat. No. 4,407,956), highvelocity ballistic penetration by small particles with the nucleic acideither within the matrix of small beads or particles, or on the surface(Klein et al. (1987) Nature 327; 70), use of pollen as vector (WO85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes carryinga T-DNA plasmid in which DNA fragments are cloned. The T-DNA plasmid istransmitted to plant cells upon infection by Agrobacterium tumefaciens,and a portion is stably integrated into the plant genome (Horsch et al.(1984) Science 233; 496; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA80; 4803). The method of introducing a nucleic acid of the presentinvention into a host cell is not critical to the instant invention.Thus, any method, e.g., including but not limited to the above examples,which provides for effective introduction of a nucleic acid into a cellor protoplast can be employed.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, activatingpromoters or selecting transformants. These cells can optionally becultured into transgenic plants. Plant regeneration from culturedprotoplasts is described in Evans et al. (1983) “Protoplast Isolationand Culture,” Handbook of Plant Cell Cultures 1, 124-176 (MacMillanPublishing Co., N.Y.; Davey (1983) “Recent Developments in the Cultureand Regeneration of Plant Protoplasts,” Protoplasts, pp. 12-29,(Birkhauser, Basel); Dale (1983) “Protoplast Culture and PlantRegeneration of Cereals and Other Recalcitrant Crops,” Protoplasts pp.31-41, (Birkhauser, Basel); Binding (1985) “Regeneration of Plants,”Plant Protoplasts, pp. 21-73, (CRC Press, Boca Raton).

The present invention also relates to the production of transgenicorganisms, which may be bacteria, yeast, fungi, or plants, transducedwith the nucleic acids, e.g., cloned QTL of the invention. A thoroughdiscussion of techniques relevant to bacteria, unicellular eukaryotesand cell culture may be found in references enumerated above and arebriefly outlined as follows. Several well-known methods of introducingtarget nucleic acids into bacterial cells are available, any of whichmay be used in the present invention. These include: fusion of therecipient cells with bacterial protoplasts containing the DNA, treatmentof the cells with liposomes containing the DNA, electroporation,projectile bombardment (biolistics), carbon fiber delivery, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, a plethoraof kits are commercially available for the purification of plasmids frombacteria. For their proper use, follow the manufacturer's instructions(see, for example, EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect plant cells or incorporated intoAgrobacterium tumefaciens related vectors to infect plants. Typicalvectors contain transcription and translation terminators, transcriptionand translation initiation sequences, and promoters useful forregulation of the expression of the particular target nucleic acid. Thevectors optionally comprise generic expression cassettes containing atleast one independent terminator sequence, sequences permittingreplication of the cassette in eukaryotes, or prokaryotes, or both,(e.g., shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith(1979) Gene 8:81; Roberts et al. (1987) Nature 328:731; Schneider et al.(1995) Protein Expr. Purif 6435:10; Ausubel, Sambrook, Berger (allsupra). A catalogue of Bacteria and Bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson et al. (1992) Recombinant DNA, Second Edition,Scientific American Books, N.Y.

Transforming Nucleic Acids into Plants.

Embodiments of the present invention pertain to the production oftransgenic plants comprising the cloned nucleic acids, e.g., chromosomeintervals, isolated ORFs, and cDNAs associated with QTLs, of theinvention. Techniques for transforming plant cells with nucleic acidsare generally available and can be adapted to the invention by the useof nucleic acids encoding or corresponding to QTL, QTL homologs,isolated chromosome intervals, and the like. In addition to Berger,Ausubel and Sambrook, useful general references for plant cell cloning,culture and regeneration include Jones (ed) (1995) Plant Gene Transferand Expression Protocols—Methods in Molecular Biology, Volume 49 HumanaPress Towata N.J.; Payne et al. (1992) Plant Cell and Tissue Culture inLiquid Systems John Wiley & Sons, Inc. New York, N.Y. (Payne); andGamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg N.Y.) (Gamborg). A variety of cell culture media aredescribed in Atlas and Parks (eds) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, Fla. (Atlas). Additional information forplant cell culture is found in available commercial literature such asthe Life Science Research Cell Culture Catalogue (1998) fromSigma-Aldrich, Inc (St Louis, Mo.) (Sigma-LSRCCC) and, e.g., the PlantCulture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (StLouis, Mo.) (Sigma-PCCS). Additional details regarding plant cellculture are found in Croy, (ed.) (1993) Plant Molecular Biology BiosScientific Publishers, Oxford, U.K.

The nucleic acid constructs of the invention, e.g., plasmids, cosmids,artificial chromosomes, DNA and RNA polynucleotides, are introduced intoplant cells, either in culture or in the organs of a plant by a varietyof conventional techniques. Where the sequence is expressed, thesequence is optionally combined with transcriptional and translationalinitiation regulatory sequences which direct the transcription ortranslation of the sequence from the exogenous DNA in the intendedtissues of the transformed plant.

Isolated nucleic acid acids of the present invention can be introducedinto plants according to any of a variety of techniques known in theart. Techniques for transforming a wide variety of higher plant speciesare well known and described in the technical, scientific, and patentliterature. See, for example, Weising et al. (1988) Ann. Rev. Genet.22:421-477.

The DNA constructs of the invention, for example plasmids, cosmids,phage, naked or variously conjugated-DNA polynucleotides, (e.g.,polylysine-conjugated DNA, peptide-conjugated DNA, liposome-conjugatedDNA, etc.), or artificial chromosomes, can be introduced directly intothe genomic DNA of the plant cell using techniques such aselectroporation and microinjection of plant cell protoplasts, or the DNAconstructs can be introduced directly to plant cells using ballisticmethods, such as DNA particle bombardment.

Microinjection techniques for injecting e.g., cells, embryos, callus andprotoplasts, are known in the art and well described in the scientificand patent literature. For example, a number of methods are described inJones (ed) (1995) Plant Gene Transfer and Expression Protocols—Methodsin Molecular Biology, Volume 49 Humana Press Towata N.J., as well as inthe other references noted herein and available in the literature.

For example, the introduction of DNA constructs using polyethyleneglycol precipitation is described in Paszkowski, et al., EMBO J. 3:2717(1984). Electroporation techniques are described in Fromm, et al., Proc.Nat'l. Acad. Sci. USA 82:5824 (1985). Ballistic transformationtechniques are described in Klein, et al., Nature 327:70-73 (1987).Additional details are found in Jones (1995) and Gamborg and Phillips(1995), supra, and in U.S. Pat. No. 5,990,387.

Alternatively, and in some cases preferably, Agrobacterium mediatedtransformation is employed to generate transgenic plants.Agrobacterium-mediated transformation techniques, including disarmingand use of binary vectors, are also well described in the scientificliterature. See, for example Horsch, et al. (1984) Science 233:496; andFraley et al. (1984) Proc. Nat'l. Acad. Sci. USA 80:4803 and recentlyreviewed in Hansen and Chilton (1998) Current Topics in Microbiology240:22 and Das (1998) Subcellular Biochemistry 29: Plant MicrobeInteractions pp 343-363.

The DNA constructs may be combined with suitable T-DNA flanking regionsand introduced into a conventional Agrobacterium tumefaciens hostvector. The virulence functions of the Agrobacterium tumefaciens hostwill direct the insertion of the construct and adjacent marker into theplant cell DNA when the cell is infected by the bacteria. See, U.S. Pat.No. 5,591,616. Although Agrobacterium is useful primarily in dicots,certain monocots can be transformed by Agrobacterium. For instance,Agrobacterium transformation of maize is described in U.S. Pat. No.5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller (1987) In: Genetic Engineering, vol. 6, P W JRigby, Ed., London, Academic Press; and Lichtenstein; C. P., and Draper(1985) In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press;WO 88/02405, published Apr. 7, 1988, describes the use of A. rhizogenesstrain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 orpARC16 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al.(1984) Plant Cell Physiol. 25:1353), (3) the vortexing method (see,e.g., Kindle (1990) Proc. Natl. Acad. Sci., (USA) 87:1228.

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al. (1983) Methods in Enzymology,101:433; D. Hess (1987) Intern Rev. Cytol. 107:367; Luo et al. (1988)Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as described by Pena et al. (1987) Nature 325:274. DNA can also beinjected directly into the cells of immature embryos and the desiccatedembryos rehydrated as described by Neuhaus et al. (1987) Theor. Appl.Genet. 75:30; and Benbrook et al. (1986) in Proceedings Bio ExpoButterworth, Stoneham, Mass., pp. 27-54. A variety of plant viruses thatcan be employed as vectors are known in the art and include cauliflowermosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaicvirus.

Regeneration of Transgenic Plants

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans et al. (1983) Protoplasts Isolationand Culture, Handbook of Plant Cell Culture pp. 124-176, MacmillianPublishing Company, New York; and Binding (1985) Regeneration of Plants,Plant Protoplasts pp. 21-73, CRC Press, Boca Raton. Regeneration canalso be obtained from plant callus, explants, somatic embryos (Dandekaret al. (1989) J. Tissue Cult. Meth. 12:145; McGranahan, et al. (1990)Plant Cell Rep. 8:512) organs, or parts thereof. Such regenerationtechniques are described generally in Klee et al. (1987)., Ann. Rev. ofPlant Phys. 38:467-486. Additional details are found in Payne (1992) andJones (1995), both supra, and Weissbach and Weissbach, eds. (1988)Methods for Plant Molecular Biology Academic Press, Inc., San Diego,Calif. This regeneration and growth process includes the steps ofselection of transformant cells and shoots, rooting the transformantshoots and growth of the plantlets in soil. These methods are adapted tothe invention to produce transgenic plants bearing QTLs and other genesisolated according to the methods of the invention.

In addition, the regeneration of plants containing the polynucleotide ofthe present invention and introduced by Agrobacterium into cells of leafexplants can be achieved as described by Horsch et al. (1985) Science227:1229-1231. In this procedure, transformants are grown in thepresence of a selection agent and in a medium that induces theregeneration of shoots in the plant species being transformed asdescribed by Fraley et al. (1983) Proc. Natl. Acad. Sci. (U.S.A.)80:4803. This procedure typically produces shoots within two to fourweeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

Preferred plants for the transformation and expression of Sclerotiniaresistance associated QTL and other nucleic acids identified and clonedaccording to the present invention include agronomically andhorticulturally important species. Such species include primarilydicots, e.g., of the families: Leguminosae (including pea, beans,lentil, peanut, yam bean, cowpeas, velvet beans, soybean, clover,alfalfa, lupine, vetch, lotus, sweet clover, wisteria, and sweetpea);and, Compositae (the largest family of vascular plants, including atleast 1,000 genera, including important commercial crops such assunflower).

Additionally, preferred targets for modification with the nucleic acidsof the invention, as well as those specified above, plants from thegenera: Allium, Apium, Arachis, Brassica, Capsicum, Cicer, Cucumis,Curcubita, Daucus, Fagopyrum, Glycine, Helianthus, Lactuca, Lens,Lycopersicon, Medicago, Pisum, Phaseolus, Solanum, Trifolium, Vigna, andmany others.

Common crop plants which are targets of the present invention includesoybean, sunflower, canola, peas, beans, lentils, peanuts, yam beans,cowpeas, velvet beans, clover, alfalfa, lupine, vetch, sweet clover,sweetpea, field pea, fava bean, broccoli, brussel sprouts, cabbage,cauliflower, kale, kohlrabi, celery, lettuce, carrot, onion, pepper,potato, eggplant, and tomato.

In construction of recombinant expression cassettes of the invention,which include, for example, helper plasmids comprising virulencefunctions, and plasmids or viruses comprising exogenous DNA sequencessuch as structural genes, a plant promoter fragment is optionallyemployed which directs expression of a nucleic acid in any or alltissues of a regenerated plant. Examples of constitutive promotersinclude the cauliflower mosaic virus (CaMV) 35S transcription initiationregion, the 1′- or 2′-promoter derived from T-DNA of Agrobacteriumtumefaciens, and other transcription initiation regions from variousplant genes known to those of skill. Alternatively, the plant promotermay direct expression of the polynucleotide of the invention in aspecific tissue (tissue-specific promoters) or may be otherwise undermore precise environmental control (inducible promoters). Examples oftissue-specific promoters under developmental control include promotersthat initiate transcription only in certain tissues, such as fruit,seeds, or flowers.

Any of a number of promoters which direct transcription in plant cellscan be suitable. The promoter can be either constitutive or inducible.In addition to the promoters noted above, promoters of bacterial originwhich operate in plants include the octopine synthase promoter, thenopaline synthase promoter and other promoters derived from native Tiplasmids. See, Herrara-Estrella et al. (1983), Nature, 303:209. Viralpromoters include the 35S and 19S RNA promoters of cauliflower mosaicvirus. See, Odell et al. (1985) Nature, 313:810. Other plant promotersinclude the ribulose-1,3-bisphosphate carboxylase small subunit promoterand the phaseolin promoter. The promoter sequence from the E8 gene andother genes may also be used. The isolation and sequence of the E8promoter is described in detail in Deikman and Fischer (1988) EMBO J.7:3315. Many other promoters are in current use and can be coupled to anexogenous DNA sequence to direct expression of the nucleic acid.

If expression of a polypeptide, including those encoded by QTL or othernucleic acids correlating with phenotypic traits of the presentinvention, is desired, a polyadenylation region at the 3′-end of thecoding region is typically included. The polyadenylation region can bederived from the natural gene, from a variety of other plant genes, orfrom, e.g., T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions)from genes encoding expression products and transgenes of the inventionwill typically include a nucleic acid subsequence, a marker gene whichconfers a selectable, or alternatively, a screenable, phenotype on plantcells. For example, the marker may encode biocide tolerance,particularly antibiotic tolerance, such as tolerance to kanamycin, G418,bleomycin, hygromycin, or herbicide tolerance, such as tolerance tochlorosluforon, or phosphinothricin (the active ingredient in theherbicides bialaphos or Basta). See, e.g., Padgette et al. (1996) In:Herbicide-Resistant Crops (Duke, ed.), pp 53-84, CRC Lewis Publishers,Boca Raton (“Padgette, 1996”). For example, crop selectivity to specificherbicides can be conferred by engineering genes into crops which encodeappropriate herbicide metabolizing enzymes from other organisms, such asmicrobes. See, Vasil (1996) In: Herbicide-Resistant Crops (Duke, ed.),pp 85-91, CRC Lewis Publishers, Boca Raton) (“Vasil”, 1996).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype. Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof the invention, provided that these parts comprise the introducednucleic acid sequences.

Transgenic plants expressing a polynucleotide of the present inventioncan be screened for transmission of the nucleic acid of the presentinvention by, for example, standard immunoblot and DNA detectiontechniques. Expression at the RNA level can be determined initially toidentify and quantitate expression-positive plants. Standard techniquesfor RNA analysis can be employed and include PCR amplification assaysusing oligonucleotide primers designed to amplify only the heterologousRNA templates and solution hybridization assays using heterologousnucleic acid-specific probes. The RNA-positive plants can then analyzedfor protein expression by Western immunoblot analysis using thespecifically reactive antibodies of the present invention. In addition,in situ hybridization and immunocytochemistry according to standardprotocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

A preferred embodiment is a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

High Throughput Screening

In one aspect of the invention, the determination of genetic markeralleles is performed by high throughput screening. High throughputscreening involves providing a library of genetic markers, e.g., RFLPs,AFLPs, isozymes, specific alleles and variable sequences, including SSR.Such libraries are then screened against plant genomes to generate a“fingerprint” for each plant under consideration. In some cases apartial fingerprint comprising a sub-portion of the markers is generatedin an area of interest. Once the genetic marker alleles of a plant havebeen identified, the correspondence between one or several of the markeralleles and a desired phenotypic trait is determined through statisticalassociations based on the methods of this invention.

High throughput screening can be performed in many different formats.Hybridization can take place in a 96-, 324-, or a 1524-well format or ina matrix on a silicon chip or other format.

In one commonly used format, a dot blot apparatus is used to depositsamples of fragmented and denatured genomic DNA on a nylon ornitrocellulose membrane. After cross-linking the nucleic acid to themembrane, either through exposure to ultra-violet light or by heat, themembrane is incubated with a labeled hybridization probe. The labels areincorporated into the nucleic acid probes by any of a number of meanswell-known in the art. The membranes are washed to remove non-hybridizedprobes and the association of the label with the target nucleic acidsequence is determined.

A number of well-known robotic systems have been developed for highthroughput screening, particularly in a 96 well format. These systemsinclude automated workstations like the automated synthesis apparatusdeveloped by Takeda Chemical Industries, LTD. (Osaka, Japan) and manyrobotic systems utilizing robotic arms (Zymate II, Zymark Corporation,Hopkinton, Mass.; ORCA™, Beckman Coulter, Fullerton Calif.). Any of theabove devices are suitable for use with the present invention. Thenature and implementation of modifications to these devices (if any) sothat they can operate as discussed herein will be apparent to personsskilled in the relevant art.

In addition, high throughput screening systems themselves arecommercially available (see, e.g., Zymark Corp., Hopkinton, Mass.; AirTechnical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton,Calif.; Precision Systems, Inc., Natick, Mass., etc.). These systemstypically automate entire procedures including all sample and reagentpipetting, liquid dispensing, timed incubations, and final readings ofthe microplate or membrane in detector(s) appropriate for the assay.These configurable systems provide high throughput and rapid start up aswell as a high degree of flexibility and customization. Themanufacturers of such systems provide detailed protocols for the use oftheir products in high throughput applications.

In one variation of the invention, solid phase arrays are adapted forthe rapid and specific detection of multiple polymorphic nucleotides.Typically, a nucleic acid probe is linked to a solid support and atarget nucleic acid is hybridized to the probe. Either the probe, or thetarget, or both, can be labeled, typically with a fluorophore. If thetarget is labeled, hybridization is evaluated by detecting boundfluorescence. If the probe is labeled, hybridization is typicallydetected by quenching of the label by the bound nucleic acid. If boththe probe and the target are labeled, detection of hybridization istypically performed by monitoring a color shift resulting from proximityof the two bound labels.

In one embodiment, an array of probes are synthesized on a solidsupport. Using chip masking technologies and photoprotective chemistry,it is possible to generate ordered arrays of nucleic acid probes. Thesearrays, which are known, e.g., as “DNA chips” or as very large scaleimmobilized polymer arrays (VLSIPS™ arrays) can include millions ofdefined probe regions on a substrate having an area of about 1 cm² toseveral cm².

In another embodiment, capillary electrophoresis is used to analyzepolymorphism. This technique works best when the polymorphism is basedon size, for example, AFLP and SSR. This technique is described indetail in U.S. Pat. Nos. 5,534,123 and 5,728,282. Briefly, capillaryelectrophoresis tubes are filled with the separation matrix. Theseparation matrix contains hydroxyethyl cellulose, urea and optionallyformamide. The AFLP or SSR samples are loaded onto the capillary tubeand electorphoresed. Because of the small amount of sample andseparation matrix required by capillary electrophoresis, the run timesare very short. The molecular sizes and therefore, the number ofnucleotides present in the nucleic acid sample is determined bytechniques described herein. In a high throughput format, many capillarytubes are placed in a capillary electrophoresis apparatus. The samplesare loaded onto the tubes and electrophoresis of the samples is runsimultaneously. See, Mathies and Huang, (1992) Nature 359:167.

Integrated Systems

Because of the great number of possible combinations present in onearray, in one aspect of the invention, an integrated system such as acomputer, software corresponding to the statistical models of theinvention, and data sets corresponding to genetic markers and phenotypicvalues, facilitates mapping of phenotypic traits, including QTLs. Thephrase “integrated system” in the context of this invention refers to asystem in which data entering a computer corresponds to physical objectsor processes external to the computer, e.g., nucleic acid sequencehybridization, and a process that, within a computer, causes a physicaltransformation of the input signals to different output signals. Inother words, the input data, e.g., hybridization on a specific region ofan array is transformed to output data, e.g., the identification of thesequence hybridized. The process within the computer is a set ofinstructions, or “program,” by which positive hybridization signals arerecognized by the integrated system and attributed to individual samplesas a genotype. Additional programs correlate the genotype, and moreparticularly in the methods of the invention, the haplotype, ofindividual samples with phenotypic values, e.g., using the HAPLO-IM⁺,HAPLO-MQM, and/or HAPLO-MQM⁺ models of the invention. For example, theprograms JoinMap® and MapQTL® are particularly suited to this type ofanalysis and can be extended to include the HAPLO-IM⁺, HAPLO-MQM, and/orHAPLO-MQM+ models of the invention. In addition there are numerous e.g.,C/C++ programs for computing, Delphi and/or Java programs for GUIinterfaces, and Active X applications (e.g., Olectra Chart and TrueWevChart) for charting tools. Other useful software tools in the contextof the integrated systems of the invention include statistical packagessuch as SAS, Genstat, and S-Plus. Furthermore additional programminglanguages such as Fortran and the like are also suitably employed in theintegrated systems of the invention.

In one aspect, the invention provides an integrated system comprising acomputer or computer readable medium comprising a database with at leastone data set that corresponds to genotypes for genetic markers. Thesystem also includes a user interface allowing a user to selectivelyview one or more databases. In addition, standard text manipulationsoftware such as word processing software (e.g., Microsoft Word™ orCorel Wordperfect™) and database or spreadsheet software (e.g.,spreadsheet software such as Microsoft Excel™, Corel Quattro Pro™, ordatabase programs such as Microsoft Access™ or Paradox™) can be used inconjunction with a user interface (e.g., a GUI in a standard operatingsystem such as a Windows, Macintosh or Linux system) to manipulatestrings of characters.

The invention also provides integrated systems for sample manipulationincorporating robotic devices as previously described. A robotic liquidcontrol armature for transferring solutions (e.g., plant cell extracts)from a source to a destination, e.g., from a microtiter plate to anarray substrate, is optionally operably linked to the digital computer(or to an additional computer in the integrated system). An input devicefor entering data to the digital computer to control high throughputliquid transfer by the robotic liquid control armature and, optionally,to control transfer by the armature to the solid support is commonly afeature of the integrated system.

Integrated systems for genetic marker analysis of the present inventiontypically include a digital computer with one or more of high-throughputliquid control software, image analysis software, data interpretationsoftware, a robotic liquid control armature for transferring solutionsfrom a source to a destination operably linked to the digital computer,an input device (e.g., a computer keyboard) for entering data to thedigital computer to control high throughput liquid transfer by therobotic liquid control armature and, optionally, an image scanner fordigitizing label signals from labeled probes hybridized, e.g., toexpression products on a solid support operably linked to the digitalcomputer. The image scanner interfaces with the image analysis softwareto provide a measurement of, e.g., differentiating nucleic acid probelabel intensity upon hybridization to an arrayed sample nucleic acidpopulation, where the probe label intensity measurement is interpretedby the data interpretation software to show whether, and to what degree,the labeled probe hybridizes to a label. The data so derived is thencorrelated with phenotypic values using the statistical models of thepresent invention, to determine the correspondence between phenotype andgenotype(s) for genetic markers, thereby, assigning chromosomallocations.

Optical images, e.g., hybridization patterns viewed (and, optionally,recorded) by a camera or other recording device (e.g., a photodiode anddata storage device) are optionally further processed in any of theembodiments herein, e.g., by digitizing the image and/or storing andanalyzing the image on a computer. A variety of commercially availableperipheral equipment and software is available for digitizing, storingand analyzing a digitized video or digitized optical image, e.g., usingPC (Intel x86 or pentium chip-compatible DOS™, OS2™ WINDOWS™, WINDOWSNT™ or WINDOWS95™ based machines), MACINTOSH™, LINUX, or UNIX based(e.g., SUN™ work station) computers.

EXAMPLES

The following experimental methods and results provide additionaldetails regarding specific aspects of protocols and procedures relevantto the practice of the present invention. The examples, which areprovided without limitation to illustrate the claimed invention, involvethe application of protocols well-known to those of skill in the art,and detailed in the references cited herein.

Mapping Population

A population of 264 recombinant inbred lines (RIL) was derived bysingle-seed-descent inbreeding from the F₂ to the F_(6:7) generationbetween a parental line resistant (P^(R)) to Sclerotinia stem rot and aparental line susceptible (P^(S)) to Sclerotinia stem rot.

Molecular Marker Analysis

DNA Isolation.

Soybean DNA was extracted using a variation of the CTAB method (Murrayand Thompson (1980) Nucl Acid Res 8:4321-4325; Keim et al. (1988)Soybean Genet Newslett 15:150-152), with the following modifications:lyophilized tissue (750 mg) was powdered by adding 2.5 g glass beads ina 50-ml tube and shaking in a paint-can shaker. The concentration ofCTAB (hexadecyltrimethyl-ammonium bromide) in the extraction andprecipitation buffers was reduced from 1% to 0.5%. After precipitationof the DNA with CTAB, the DNA pellet was dissolved in 2 ml 1 M NaCl byshaking at 65° C., 200 rpm, for approximately 2-3 h. The DNA wasre-precipitated by adding 4.5 ml ice-cold 95% ethanol. The precipitatedDNA was then washed with 1 ml of 65% ethanol, and again with 1 ml of 85%ethanol, to further remove salts. After the ethanol washes, the DNA wasdissolved in Tris-EDTA buffer (10 mM:1 mM) at a concentration of 500ng/μl, and stored at 20° C.

Restriction Fragment Length Polymorphism (RFLP) Analysis.

Most of the RFLP markers utilized were from PstI-cloned genomiclibraries, and were either public (Keim and Shoemaker (1988) SoybeanGenet Newslett 15:147-148) or proprietary to Pioneer Hi-Bred Intl., Inc.(denoted by the prefix: PRP). Some RFLP markers corresponded to USDA-ARS(Beltsville, Md.) cDNA clones (prefixed pBLT). The cloned inserts usedas probes were amplified by the polymerase chain reaction with T3 and T7primers. The restriction enzymes EcoRI, HindIII, EcoRV, DraI, TaqI, andHaeIII were used to digest soybean parental and population DNA.Restriction enzyme digestion of DNA, electrophoresis, Southern transfers(blotting), and DNA hybridization were all performed as describedpreviously (Keim et al. (1989) Theor Appl Genet. 77:786-792).

Amplified Fragment Length Polymorphism (AFLP) Analysis.

The KeyGene protocol was used in the AFLP amplification with thefollowing modifications. Briefly, DNA was digested with EcoRIIMseIrestriction enzymes and ligated to EcoRI and MseI adaptors. Therestriction ligation (RL) products were diluted 1/10 for use in the Plus1 reaction. The Plus 1 reactions contained 5.0 μl of the diluted RLproducts, 1.5 μl each of the Mse+1 primer and Eco+1 primer (50 ng/μl),0.4 μl of a mixture of dNTP's (25 mM), 5.0 μl of 10× high magnesium HotTub buffer, 0.2 μl Hot Tub polymerase (3 units/μl), and 36.4 μl of H₂Ofor a final volume of 50.0 μl. The Plus 1 amplification used twentycycles of 94° C. (30 sec.), 56° C. (1 min.), and 72° C. (1 min.). ThePlus 1 products were diluted 1/20 for use in the Plus 3 reactions. ThePlus 3 reactions contained 5.0 μl of the diluted Plus 1 products, 0.6 μlof the Mse+3 primer and 0.5 μl γ³³P-ATP labled Eco+3 primer (10 ng/μl),0.2 μl of a mixture of dNTP's (25 mM), 2.0 μl of 10× high magnesium HotTub buffer, 0.1 μl Hot Tub polymerase (3 units/μl), and 11.5 μl of HPLCH₂O for a final volume of 20.0 μl. The Plus 3 amplifications used a 94°C. (2 min.), followed by 13 cycles of 94° C. (30 sec.), 65° C. (30 sec.,decrease annealing temp by 0.7° C. each cycle), and 72° C. (1 min.)followed by 23 cycles of 94° C. (30 sec.), 56° C. (30 sec.), and 72° C.(1 min.), and followed by 72° C. (2 min.) step. The Plus 1 and Plus 3reactions differ form the reactions in KeyGene's protocol in the use ofHot Tub polymerase instead of Taq polymerase. The Plus 3 products wereloaded to polyacrylamide gels (4.5% acrylamide, 7 M urea, 0.5×TBE), andthe gel was run for 3 hours at a setting of 45° C., 120 Watts. Thelabeled products separated on the Plus 3 gels were detected using aphospho-imaging system.

Simple Sequence Repeat (SSR) Analysis.

SSR markers used were developed by either public sources (Cregan et al.(1994) Meth Mol Cell Biol 5:49-61) or by DuPont. Amplification by PCRwas performed in 10 μl reaction volume containing 10 ng of genomic DNA,1× reaction buffer, 1.5 mM MgCl₂, 0.2 mM dNTP, 0.34 μM of forward andreverse primers, and 0.5 units of AmpliTag DNA polymerase (Perkin-ElmerCetus, Norwalk, Conn.). The SSR PCR products were analyzed either bysize separation on 2% metaphor gel and visualized by ethidium bromidestaining, or by sequencing on an automated ABI 377 sequencer usingGeneScan software.

Sclerotinia Screening

Inoculum Source.

Sclerotia from infected greenhouse plants were stored in culture tubesin a 45° C. refrigerator. Six days before plant inoculation, sclerotiawere plated on potato dextrose agar (PDA) media and placed in a 25-27°C. incubator. After 4 days, a single plate with uniform mycelial growthextending to at least 1½ inches in diameter was used. Plugs were cut in4 mm square from the edge of the mycelia and were plated in the centerof PDA plates. Plates were put back in the 25-27° C. incubator for 24hrs.

Carrot Preparation.

Large fresh carrots were rinsed in distilled water and peeled. Carrotswere cut into 5-10 cm sections and then pushed lengthwise through aZyliss brand French frier. The resulting 8 mm square lengths were cutinto approximately 2 mm thick pieces. Carrot pieces were placed in anautoclave bag and autoclaved for 1½ to 2 minutes.

Carrot Inoculation.

Autoclaved carrot pieces were placed on the edge of the growing myceliathat had incubated for 24 hrs. Eight to twelve pieces were placed oneach plate, depending on the mycelial diameter. Each carrot piece wasplaced with its outer edge positioned on the growing edge of themycelia. Plates were returned to incubator for 22-24 hrs.

Stem Inoculation.

Plants were inoculated between V3 and V4 stages. Each inoculated carrotpiece was placed on a 2 inch square parafilm just at the tip of thecenter slit. The carrot piece on the parafilm was positioned on the steminternode just above the petiole immediately below the last fullydeveloped leaf. The parafilm was wrapped around the stem with the centercut positioned downward. The carrot piece was pulled into contact withthe stem while wrapping the parafilm.

Plant Incubation and Removal of Inoculum.

Inoculated plants were placed on a shaded bench where a humidifier runscontinually. The humidity in the room was set at 70%. Plants stayed inthis humidity chamber for 48 hrs. Inoculum was removed form steminoculated plants by taking off parafilm and pulling carrot pieces off.Paper tissues were used to wipe the plant stem of remaining inoculum.

Disease Scoring.

Plants were rated for disease severity 6-8 days after inoculation.Plants were rated on a scale of 1 to 9, with 1 being stem girdled andhaving large lesion, 3 being stem girdled, 5 being larger lesion inlength, 7 being small lesion and black margin, and 9 being no infectionor browning. The final score for each RIL was an average of tworeplications and expressed as the percentage of the P^(R) score.

Genetic Mapping

Genetic mapping and QTL analysis were performed using MapManager QTb27(Manly (1993) Mamm Genome 1:123-126; Manly and Olson (1999) Mamm Genome10:327-334). The Kosambi centiMorgan function was used. A QTL wasdeclared if its Likelihood Ratio Statistic (LRS) exceeds the thresholdof 12.3. The threshold was established by performing 500 permutationtests. Composite Interval Mapping (CIM) were also performed by addingbackground loci (usual the loci with the largest effect) to simpleinterval mapping (SIM).

QTL Mapping

Genetic Mapping

Genetic mapping has placed 333 molecular markers to 20 linkage groups(Lg) that are corresponding to 20 soybean chromosomes and public linkagegroup nomenclature. Of 331 markers, 53 are RFLP, 159 are AFLP, 21 DuPontSSR and 100 public SSR. The linkage map covers 2400 cM.

QTL Mapping

QTL analysis for stem inoculation data identified 7 QTL responsible forwhite mold resistance. The QTL with larger effects on white mold locateon soybean Lg A1, D2, and L. With Composite Interval Mapping, the QTL onLg A2, B2 and D2 show additive effects, and these three QTL togetherexplain 42% of total variation.

Specific Linkage Groups

QTL on Lg A1

This QTL has LRS of 19.0 and explains 14.0% of the total variation. Thefavorable allele (allele which is resistant to Sclerotinia stem rot) ofthis QTL comes from P^(S). The QTL is in the interval ofSatt155-SLS1C.L24 with a distance of 1.3 cM. It is interesting thatSLS1C.L24 is a homolog to Berberine-bridge forming enzyme (BBE). The BBE[9S0-reticuline:oxygen oxidoreductase (methylene-bridge-forming), EC1.5.3.9] is a key covalently flavinylated oxidase in thebenzophenanthridine alkaloid biosynthesis in plants (Kutchan andDittrich (1995) J Biol Chem 270:24475-24481; Blechert et al. (1992) ProcNatl Acad Sci USA 92:4099-4105; Dittrich and Kutchan (1991) Proc NatlAcad Sci USA 88:9969-9973; Chou and Kutchan (1998) The Plant J15:289-300). The alkaloid families have pharmacological activities.Berbrine, for example, is currently used as an antibacterial treatmentfor eye infection in Europe and for intestinal infections in the FarEast. The benzophenanthridine alkaloid sanguinarine is an antimicrobialused in the treatment of periodontal disease in both the United Statesand Europe (Kutchan and Dittrich (1995) J Biol Chem 270:24475-24481). Inaddition, BBE has anti-phytophthora and anti-Pythium activity, andcarbohydrate oxidase activity (Stuiver et al. (1998) WO 98/13478). TheBBE-transgenic plants may have enhanced resistance to pathogens (Stuiveret al., 1998). BBE and several other enzymes in the pathway were inducedby elicitors in California poppy (Blechert et al. (1992) Proc Natl AcadSci USA 92:4099-4105; Dittrich and Kutchan (1991) Proc Natl Acad Sci USA88:9969-9973). The benzophenanthridine alkaloids accumulated in thesuspension cells of plants in response to the addition of elicitors(Schumacher et al. (1987) Plant Cell Rep 6:410-413; Cline and Coscia(1988) Plant Physiol 86:0161-0165; Eilert et al. (1984) J. Plant Physiol119:65-76). The alkaloid pathways may also be regulated byoctadecanoic-derived components and jasmonic acid (Blechert et al.(1992) Proc Natl Acad Sci USA 92:4099-4105; Kutchan (1993) J. PlantPhysiol 142:502-505; Facchini et al. (1996) Plant Physiol 111:687-697).It is not known if the benzophenanthridine alkaloid biosynthesis pathwayexists in soybean, but its potential antifungal activity andcarbohydrate oxidase activity imply that it could be involved inSclerotinia stem rot resistance.

QTL on Lg A2

This QTL has LRS of 19.3 and explains 12.0% of the total variation. Thefavorable allele of this QTL comes from P^(S). The support interval ofthis QTL is Sat129-Satt329 of 31.7 cM.

QTL on Lg B2

This QTL has LRS of 18.9 and explains 12.0% of the total variation. Thefavorable allele of this QTL comes from P^(R). The support interval ofthis QTL is Satt556 and P1694 of 12.7 cM.

QTL on Lg D2

The QTL is in the interval of PHP8701R-Satt311 of 14.5 cM. This QTLexplains 14.0% of the total variation with LRS of 26.6. The favorableallele of this QTL comes from P^(R).

QTL on Lg E

The support interval for this QTL is the PHP100771-PHP8241O with thegenetic distance of 30.8 cM. The peak interval is in thePHP10118C-Satt231 of 18.8 cM. This QTL has a LRS of 24.0, and explains10% of the total phenotypic variation. The favorable allele of this QTLattributed to P^(S).

QTL on Lg J

The QTL is in the interval of P1047-A724_(—)1 of 22.5 cM. The QTLexplains 6% of phenotypic variation with the LRS of 13.6.

QTL on Lg L

This QTL is in the interval of Satt523 and SLS2C.F20 with the distanceof 11.7 cM. This QTL explains 16.0% of the variation with the LRS of26.1. Interestingly, SLS2C.F20 is an EST coding for pathogenisis-relatedprotein 1 (PR1). The significance of PR 1 proteins lies in the fact thatthey show strong antifungal and other antimicrobial activity. Alexanderet al. (1993) Proc Natl Acad Sci USA 90:7327-7331, reported thattransgenic N. tabacum cv Xanthi nc, which constitutively produce PR-1aprotein, showed an increased tolerance to two oomycete pathogens.Niderman et al. (1995) Plant Physiol 108:17-27, tested the antifungalactivity of purified tobacco and tomato PR-1 proteins againstPhythopthora infestans. The basic PR-1 proteins (PR-1 g (tobacco) andP14C (tomato), found to be most effective in the assays. Therefore,there is a possibility that The EST, SLS2C.F20, is involved in theSclerotinia stem rot resistance.

Table 2 summarizes the chromosome intervals, and subintervals defined bythe flanking markers associated with Sclerotinia stem rot resistance.Subintervals are not given for linkage group A1 because it is alreadytightly defined to a region of 1.3 cM. Due to asymmetry of an LRS peakwithin an interval, subinterval boundaries that fall outside the definedinterval have been given a distance of 0 cM from the interval boundary,see, e.g., linkage group B2.

TABLE 2 Summary of Linkage Intervals Linkage Distance to flanking groupDefined chromosome interval Subinterval markers A1  1.3 cM flanked bySatt155 and SLS1C.L24 N/A N/A A2 31.7 cM flanked by Sat_129 and Satt32995%  7 cM to Sat_129 16 cM to Satt329 90%  5 cM to Sat_129 14 cM toSatt329 80%  1 cm to Sat_129 11 cM to Satt329 B2 12.7 cM flanked bySatt556 and P1694 95%  0 cM to Satt556  9 cM to P1694 90%  0 cM toSatt556  9 cM to P1694 D2 14.5 cM flanked by PHP8701R and Satt311 95%  0cM to P8701R 10 cM to Satt311 90%  0 cM to P8701R  8 cM to Satt311 E18.8 cM flanked by PHP10118C and Satt231 95%  7 cM to P10118C  2 cM toSatt231 90%  6 cM to P10118C  1 cM to Satt231 J 22.5 cM flanked by P1047and A724_1 95%  4 cM to P1047 15 cM to A724_1 90%  4 cM to P1047 14 cMto A724_1 L 11.7 cM flanked by Satt523 and SLS2C.F20 95%  2 cM toSatt523  4 cM to SLS2C.F20 90%  1 cM to Satt523  3 cM to SLS2C.F20

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques, methods, compositions,apparatus and systems described above may be used in variouscombinations. All publications, patents, patent applications, or otherdocuments cited in this application are incorporated by reference intheir entirety for all purposes to the same extent as if each individualpublication, patent, patent application, or other document wereindividually indicated to be incorporated by reference for all purposes.

Sequence Listings

(SLS1C.L24) SEQ ID NO: 1 ganagcgctggagctccaccgcggtggcggccgctctagaactagtggatcccccgggctgcaggaattcggcacgagGAGGGTCTCTCCTACGTTGCCAAGGATCCATTTGTCGTCCTTGACCTCATAAACCTTCGAAAAATCGAAGTGGACGCAGAAAACAGCACTGCATGGGTTCTAGCTGGTGCAACCATTGGTGAACTTTACTACAGCATTAGCCAGAAAAGCANAACACTAGGGTTTCCAGCAGGTGTGTGCCCCCCTGTGGGAACTGGTGGCCATTTCAGTGGTGGTGGCTATGGATTCTTGATGCGTAAGTTCGGTCTTGCTGCTGATAATGTGATTGATGCTCACATACTTGATGTGAANGGTAATCTTCTTGATANAGAAGCCATGGGTGAGGATCTGTTTTGGGCCATTATNAGGAGGTGGGGGAGCAANCTTTGGAGTCATCGTGGCTTGGNAGATANAACNTGTTTCAGTTCCATCAACTGTGACAGTGTTTAGGGGTTCCAAGGACATTGGGACAAAATGCAACCGAGATTGTTCATAAGNGGGAACCTN GTGGNGAATAAACTTGNGGG(SLS2C.F20) SEQ ID NO: 2 gctctagaactagtggatcccccgggctgcaggaattcggcaccagATTAAGCATAATTAATATAATGGGGTACATGTGCATTAAGATTTCGTTTTGTGTGATGTGTGTGCTGGGGTTGGTGATCGTGGGTGATGTTGCCTACGCTCAAGATTCAGCAGAAGACTACGTGAATGCACACAATGCAGCACGAGCAGAGGTGGGTTCTCAATCACCAAGACAAACAGTGATTGTTCCAAGTTTGGCTTGGGATGATACGGTTGCTGCTTATGCAGAGAGCTATGCTAATCAACGCAAAGGTGACTGCCAACTGATCCACTCTGGTGGTGAATACGGAGAGAATATTGCAATGAGCACTGGTGAACTAAGTGGCACAGATGCAGTGAAAATGTGGGTTGATGAGAAATCCAACTATGACTATGATTCTAACTCTTGTGTTGGAGGAGAGTGCCTGCACTACACACAGGTCCGTTTGGGCTAACTCGGTGCGTCTTGGATGTGCCAAAGTGACATGTGATA A (PHP8701R)SEQ ID NO: 3  CACAGTCACAGCTTAATGGCGGAGGAAGAAGTTGCGGCACCCGCTGCCAGCCCCGTTCCCCCTGATAACAAACGCAAGCTCGAAGATCTGCAACCCGAAAACGCCGAATCTAATGCCAACTCCATCTCCGACGCCGTGAACGCCGATGACGCCGCCGTTTCCGCCGAGACCGAGAACAAGCGTCTCCGCCTCGACGACCACCAGGATGGCCTCGGTACCGTTTTTGTCAATCTCAACTATTCGTGGTTTTGCAATGCTCGTAATAATAATTTATTTCATTCACTATTAGTATATAAATCCTTGAGGATGATATAGGCTTTAGGCAA (SATT155) SEQ ID NO: 24 TTAACATCGTATATATGGATTGCTTATGTGCAATGGCAGATCCAACACCTGGCCTAATTGGGGAATTTATTTTTAAAAAATATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTAAAACAAAAATTAAAATTAACCACAGTGAGCTAAAAATGGAATGAATTGTGCAGCAAAATAAATTATAAAATTACTTAATGAAATGAAAAAAAGTAAAACTTAAGAAAAAAAAAAGCTAGTGATAAAAAATTAGGAGTTGTG (SAT_129) SEQ ID NO: 25 TTTAGCGTGGAATTAAGTTTAAACTTAGGTTAAAAGAGAATAACTTAAATTAATATTCTCAAATTTAGATATTAAAAATATAATTTCTTTAAATTTGGATTAAAGAAAATTTGTGGNTCTTAGTTATTTGATAGTTTGACCTTATTTCAGTACAAGTCCGGTGAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAGAACCAACCTTTTTCTTTTGCCAAGAAAAATAATACCTTAAGTCCCGAACATGTGAAGATAACTCACAATTCTTACRCGGTTTCTAACTGATACCTCCTCCACCCGACGATATTGCTAGAAACGAGAATTTATAAACTTATGAAAATTTTATCATTAAATTAAATAATATCACTCTTTAAAATTAAATTTTGTATCAAACAATTGACATATCTCATTTGATTTTCAGATCATTATGCTAAGCGCTTCANCAAATAAATTATCCAGATTGAAAGAGCAGGCCGAGGAATACGCAGCTCTCATCATCCAAGAGTTAGACCCTGAAAG ACTTCGCTACAT (SATT329)SEQ ID NO: 26  CCGTGTTATGTCGTCNTTGGGTCAAATTGGGTGCCATGATTATGACCAATAGCTTGGCTTTCATTCAGAGTGCCTTGACTGAGCATTATCAGTAGCATGCCTTCTACCATTAAGTCACTCGAACCAGTTGCAAGTAGTTGGTCTCTACAACGAAAAATGAAGAACCATGAACGACGAAAAATGAAGAAAAATAGAACCACTAAGAACGAAAAATGGAACCAACAACAACACTTACAGCCCAGTTTCGAAATCCTTTAAATAGAGAAAATAATATCACTATTATAATTTAGGAATGTTCATGGTTATCAATTACTCACGAACTTGAATGGACGCAAAATTGGATTTAGTATTTTTTGCATTGAATCAAATCTTATCTAATTCTTTNCAANCCATNGATTTTNGGGTGNANTCACATATTTTTATNTGTCTANATATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTAAATTTTTTTAATATCAATTACTATTACTATCATAACTTAGTAAAAGTGACATTATCTTCTTTTTTTTTTCAGTTCTCACGTTTTATTCGGTTACTTTTACGAAGATTAGGATTTTAACTGCCATTTGATTTTAAAAATATCTCTTTTAATTTTTAATTAATTTATTTCAAACTATTTTAGATTTAGTTAAAGTTTTATTTGTATTACTCATTTAACTATTAGAGAACATTAGAATTTTCAAAATGCTCTAATAGTTTTAGTTTTTTAAGGCTCCGTGAGTGTGGTGATTAAATTTATTTATTATATTTCAATTTTTAAATCATGTTTAATATAATTTCATTTAGTTTCAAATATAAGCAACAAAATCCCTTACTTTTGGGTCTCAAATATAAATAAATTTTAATTACTTATGTCTTATTTAATGAAACTCTCTCAAAAATATCATTTTTTAATGAGGTCAAAGACTTTTTTATGTTTGAT (SATT556) SEQ ID NO: 27 TCCCATTGCTTCGGGCCACAAGTCTACGACTATAATAATTCGCCAAACAGAGTGTACCCAATATTTTTCAAAAGCAAACAACACAGAAGAATCACCACACATAACAAGCAATCATAACCCAGATACAGACAATAAAACCCGATAAATAAGATTTCATCAATAATAATAATAATAATAATAATAATAATAATAATAATAATACTCAAAGACCAAAATTTCATTTTCGAAACATGATATAGGCTTCAGAGATGAACGAACATAAAATACATAAGAAAACAAGGT GCACAATTAGG(SATT063: closest public marker to P1694) SEQ ID NO: 28AAAATTATAANTATAATAATGAAAATGATTAACAATGTTTATGATAATTATACATATTATATTTTTTACTGCATAGGTATTATTATTATTATTATNATTATTATTATTATTATTATTATTATTATTATTATTATTATTAAACTAATTGTTATTAACTGATGCAAGTAATAAAAAAACTANNNATCAGCGATGAANATGAAANTGCTAAAAATATTNNATACACAACTGTGANNAGGTNAGATGTAANTGNGGCNCCCTNANTCNGCTCAATGTGTAATTGNGGTCTNCTATTCCAAGANAGAGACANGNGGTCCNTCTANNTCAGAAAANNCANAATNGNGGNCCTCCATATTTATAAAAANGTGTAACCCNGCGGGNCANCNTCACCNCNATCTANGCCNCNTCNCTTNNATTNCTCACATANTAACNAACCNACCNCTCCNCGCTGNTACTTN (SATT311) SEQ ID NO: 29 ACAAAGAATTCATTTTCCTCCATTACTTGGTCTATTTTCATCTTCAATTCAGTGTTTTTCACTTCTAACATGACATTCTTCTITGTTATATCATCCTGCTAATCAAAGAAGCTAATTTTATATGTAATGAATTAATGTGGAGTAGAAAAGATAAAGATCAATAATCTATGACAAGACCGAAAAGATTGAGGTAAAATAAAATCTCACATGATAAAAGCTCAACTTAGGGGGAACCACAAAAATCTTAATCAAACAAACCACAAAAAGATGAAACAAAATAGTAATAGTAATATAATATAACAATGCCACATATATATTATACTAATAAACAATAATAATAATAATAATAATAATAATAATAATAATAATATATGTAGTAGTGAATTCATCACAGCCTGAGCTTCAACTTACAACTACTCCTAGAAATTCTAATATCATTACTAGCACCAAGATTATCCACCTCAAGCCTACACCTAACCCTGAACTTGACTTTGAAGAGCTTCAGCTTACCAAGCTTAACTCTCACAGGCTAATTCACCTTAAGATTGAGAGGAACATTGTTGGTCTGTTGCAACTACTCCTCAATTCTATTGACCAAACCACTTGCATCATGTCCTTGACCAGTTAGAGGCAAATCAAGCACTGTTGTGTTCCTATGACCCTGGTAGAACTTTGGCAAGGACCCTTCACACA(SAT_124: closest public marker to PHP10118C) SEQ ID NO: 30GGATCCATTCCACTTTTTGTACAATATTTTATGCATTAAATACTTAGAATTATATACATAATAACAGTATATTATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATTGACAGACCTATATGCGTTAGAGGTGGATAAATGTATCTCTTTATTTTAATAAAATTACATATATTTATCTTAAAATTATATAATTTATACCACTAATGGATGTTTGAACTTGTTATTGTTAGAACAAATCTCTTTTAACCTCTGATATTACAAACTACAAAGAAACACGGGTTATTAATAAAATGCGAAAATTACAGATCAAAACATACCTCCAGCCATTGCTATGAAGA (SATT231) SEQ ID NO: 31 TTATTGTTTTTTATACTGTTATATGTGCAAAATGTTCATCATCTTTTTCTGATCCCTAATTTCTGCTTGTGAAACTGGCTGATTATTTTTTATTAGAAATTAGAAATAAATGCACTTTAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAATAGTAATCTGTGATGGGAACAGAAGGAAGTTTTGATGTTGATTCGTGGCCTTTTTTTTCTTTTCATTTTTTGTCTAATTTTTAAATGAGTTACTTTTGCATGTTTTTTCTTCTCCGCTAATTTTGGTGTGGTAGATTCCTGATGTCTGTTAGAGATTTTTTTGGGGAATAAGAATAATATTTATCTGTTTTCCTCTCCTGTTGTTTAATTTTCCTATGCTTTTTTATGACTAGGTTGTAACTTTTATTTATTTATTANNTTGACAAGGTNTCAACTTTATTCTGTTTGATTTGAATCCGATTCT GGATTTTTATNC(SATT596: closest public marker to P1047) SEQ ID NO: 32GAACTCTGGTCCATCCCTTCGTCCACCAAATATTCAAATGTTCCTTTTTAGGGTTATCTTTTTCCTTCCACCATTTTTAACATCTCTTTCTCCTTTGCATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTAACCTATACCTANAACAATCCTCTATTTTAATTAAATAATCCCNCTTAGTATTCATGTCTCTCTCTCTATAGGAAACTTTACACTCGTATGATTACTATCTTGTACGGAATCGACGGGTAGTCGACACTTGTTAACTGACTATCACTTTACACATATTATTATTGTGAACAATTATCAACTCGGTCACTATCATCTTTTATAAGACAAAAGACCATGTAAGAATAGGAAAGTCATATTTTGTTTACAATATACACCTTAGACAAATATCTATATCCTTTATACAAACAAGGCTCTAACACATGCAGAAAGTACCAAAATGCCCCTAACTTTCTGAGGTGTCGTCGTCTGGGGGATTTTGTTCCTGCCCAAAATGGTTCGTGTGGGTTTTTAGAATTTCTTATGGAATTGGGATACGTGGTACGCGTACACGCGTTTTGGTGG (SATT523) SEQ ID NO: 33 GGGTCAACAAAAGTTGATCATGAAATTGTGGAGCTCATTTCGGCCAAGAAAATTAATCAACTCCAGTTAAACTTGTGTTTATCCTATAAATTGTAAAATTATTGCTTATCATGCTTATTGAAAAAATTACATCATTATCTCAATGTACTGTACATTTTTATTGTAAATTTTTTTATGTTATATTATATAGTGACACGTTTTGATATAGCTAATTTCTTCCTTGAAGAATTTTCTGCTGTAAATTTAATTGATAAAATAATAGAAAGACTTTTTTAAAGAAATTTAATAATAATAATAATAATAATAATAATAATAATAATAATAATAAGTTCTATTATTTGCGAATAAAGTTAAAAATAACAGCCGAAAAAGTTCAACAAATTTACAACTTTTGCATGTGAAAGCTACAACTTCAATTTTTCTTTTATGTAAGTCACTAAACTTACATATATTTAACAAATTTTGCAAATATATCTTGCATAAGTAAATTATAAAATAAATTTGGTTTACATGTGTTTATTAGAATAAAAATTTAAAATTAATATAATTAAATTATATTTTTATATAAAGAATGGATCAAAATTAAATTTATATTTTTATAAGTAGAGTGCTTTTATTTAAATATACATACTTTGTCAGAATGTATATGGCTACACTTCGTAAACAGACAAACTAGCACACGGTCACATATTTTTCAATTCAAAATTGTGACGATGAAGAATAAAGTATCAA

1-20. (canceled)
 21. An isolated or recombinant nucleic acid comprising:a) a polynucleotide sequence selected from: SEQ ID NO:1 (SLS1C.L24), SEQID NO:2 (SLS2C.F20), and SEQ ID NO:3 (PHP8701R); b) a polynucleotidesequence with at least about 97% sequence identity to a polynucleotidesequence of (a); or c) a polynucleotide sequence complementary to (a) or(b).
 22. The isolated or recombinant nucleic acid of claim 21, whichnucleic acid is associated with Scierotinia stem rot resistance insoybean.
 23. The isolated nucleic acid of claim 21, wherein the nucleicacid comprises a polynucleotide sequence with at least about 99%sequence identity to a polynucleotide sequence of (a) or apolynucleotide sequence complementary thereto.
 24. The isolated orrecombinant nucleic acid of claim 23, which nucleic acid is associatedwith Sclerotinia stem rot resistance in soybean.
 25. The isolated orrecombinant nucleic acid of claim 21, wherein the nucleic acid sequencecomprises the polynucleotide sequence of SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3, or a sequence complementary thereto.
 26. The isolated orrecombinant nucleic acid of claim 25, wherein the nucleic acid sequencecomprises the polynucleotide sequence of SEQ ID NO:1 or a sequencecomplementary thereto.
 27. The isolated or recombinant nucleic acid ofclaim 25, wherein the nucleic acid sequence comprises the polynucleotidesequence of SEQ ID NO:2 or a sequence complementary thereto.
 28. Theisolated or recombinant nucleic acid of claim 25, wherein the nucleicacid sequence comprises the polynucleotide sequence of SEQ ID NO:3 or asequence complementary thereto.
 29. A plant cell comprising the isolatedor recombinant nucleic acid of claim 21, wherein the isolated orrecombinant nucleic acid is operably linked to a promoter functional inthe plant.
 30. A plant comprising the plant cell of claim
 29. 31. Theplant of claim 30, wherein the plant is soybean, sunflower, canola,alfalfa, pepper, potato, or tomato.
 32. The plant of claim 30, whereinthe plant is soybean.
 33. The soybean plant of claim 32, wherein theplant shows increased resistance to Sclerotinia stem rot when comparedto a control soybean plant lacking the isolated or recombinant nucleicacid.
 34. Seed from the plant of claim 30, wherein the seed comprises anisolated or recombinant nucleic acid comprising: a) a polynucleotidesequence selected from: SEQ ID NO:1 (SLS1C.L24), SEQ ID NO:2(SLS2C.F20), and SEQ ID NO:3 (PHP8701R); b) a polynucleotide sequencewith at least about 97% sequence identity to a polynucleotide sequenceof (a); or c) a polynucleotide sequence complementary to (a) or (b). 35.The plant cell of claim 29, wherein the promoter is a 35S cauliflowermosaic virus (CaMV) promoter.
 36. The plant of claim 30, wherein thepromoter is a 35S cauliflower mosaic virus (CaMV) promoter.